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Scale keratin in lizard epidermis reveals amino acid regions homologous with avian and mammalian epidermal proteins
Abstract
Small proteins termed beta-keratins constitute the hard corneous material of reptilian scales. In order to study the cell site of synthesis of beta-keratin, an antiserum against a lizard beta-keratin of 15-16 kDa has been produced. The antiserum recognizes beta-cells of lizard epidermis and labels beta-keratin filaments using immunocytochemistry and immunoblotting. In situ hybridization using a cDNA-probe for a lizard beta-keratin mRNA labels beta-cells of the regenerating and embryonic epidermis of lizard. The mRNA is localized free in the cytoplasm or is associated with keratin filaments of beta-cells. The immunolabeling and in situ labeling suggest that synthesis and accumulation of beta-keratin are closely associated. Nuclear localization of the cDNA probe suggests that the primary transcript is similar to the cytoplasmic mRNA coding for the protein. The latter comprises a glycine-proline-rich protein of 15.5 kDa that contains 163 amino acids, in which the central amino acid region is similar to that of chick claw/feather while the head and tail regions resemble glycine-tyrosine-rich proteins of mammalian hairs. This is also confirmed by phylogenetic analysis comparing reptilian glycine-rich proteins with cytokeratins, hair keratin-associated proteins, and claw/feather keratins. It is suggested that different small glycine-rich proteins evolved from progenitor proteins present in basic (reptilian) amniotes. The evolution of these proteins originated glycine-rich proteins in scales, claws, beak of reptiles and birds, and in feathers. Some evidence suggests that at least some proteins contained within beta-keratin filaments are rich in glycine and resemble some keratin-associated proteins present in mammalian corneous derivatives. It is suggested that glycine-rich proteins with the chemical composition, immunological characteristics, and molecular weight of beta-keratins may represent the reptilian counterpart of keratin-associated proteins present in hairs, nails, hooves, and horns of mammals. These small proteins produce a hard type of corneous material due to their dense packing among cytokeratin filaments.
... For b-keratin, there are no different types of proteins [16]; the filament and matrix are incorporated into one single protein [26]. Finally, the molecular mass of a-keratin ranges from 40 to 68 kDa, which is much larger than that of b-keratin, 10-22 kDa [37]. [16]. ...
... Based on a-helical structure Based on b-pleated sheet structure Molecular mass [37] 40-68 kDa 10-22 kDa ...
... On the other hand, feathers (b-keratin) involve only one type of protein, and there are no distinct phases in the synthesis of the proteins. The proteins appear to increase in a coordinated fashion, and the detailed mechanism is poorly known [16,37,[78][79][80]]. ...
An ubiquitous biological material, keratin represents a group of insoluble, usually high-sulfur content and filament-forming proteins, constituting the bulk of epidermal appendages such as hair, nails, turtle scutes, horns, whale baleen, beaks, and feathers. These keratinous materials are formed by cells filled with keratin and are considered ‘dead tissues.’ Nevertheless, they are among the toughest biological materials, serving as a wide variety of interesting functions, e.g. scales to armor body, horns to combat aggressors, hagfish slime as defense against predators, nails and claws to increase prehension, hair and fur to protect against the environment. The vivid inspiring examples can offer useful solutions to design new structural and functional materials.
... [17][18][19][20][21] The molecular characteristics of -keratins of setae, as those of reptilian scales in general, are poorly known, but recent molecular studies have sequenced for the first time some of these -keratins. [22][23][24] The proteins in lizard scales contain a high amount of glycine and over 8% of proline, and they have been indicated as glycineproline-rich proteins. ...
... A core region of 20 amino acids containing five prolines (28% of the prolines in the whole protein), presents two strand -folds, and the specific analysis of this region has shown a high degree of homology in the corebox (average of 55%) among reptilian -keratins. 23,24 The most representative amino acids of -keratins from reptilian scales so far sequenced in comparison with an avian scale and feather keratins are presented in Table 1. All reptilian -keratins are rich in glycine (19-29%) and serine (7-17%), expecially in gecko -keratins. ...
... The predicted secondary structure has shown that most of the glycine-rich regions possess a random coil conformation and show little homology with mammalian "glycine-tyrosine-rich keratin associated proteins." 23,35 The central region (boxed in Figures 11 and 12) has high homology (60-80%) with the core region of avian -keratins, and this detail indicates that a common precursor for -keratins was probably present in stem reptiles from which reptiles and birds derived. From the ancestral proteins, a diversification probably occurred in the evolutionary lineages of snakes, lizards, turtles, crocodilians, and birds. ...
The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.
... The ultrastructural study on lizard beta-cells has shown that mRNAs for beta-keratins are present in the cytoplasm and are often associated with small and large keratin filaments. This observation suggests that newly synthesized proteins are immediately packed to form beta-keratin filaments (36,39). In lizards, the analysis of the structure of genes for betakeratins, and the presence of some labelling in the euchromatin of the nucleus of beta-cells suggest that the primary transcript is almost devoid of introns (32). ...
... The analysis of the amino acid sequence and the predicted secondary structure of some known beta-keratins from lizards, snakes and the partial sequence of the alligator are presented in Fig. 4. Different amino acid regions exhibit some identity with avian and mammalian proteins (39). These sequences show that the proteins comprise three main domains: two external domains rich in glycine-sequences and with variable identity with mammalian high glycine-tyrosine proteins (39), and a central region rich in proline with good identity to avian scale and feather keratins (Figs 3c, 4 and 5a). ...
... The analysis of the amino acid sequence and the predicted secondary structure of some known beta-keratins from lizards, snakes and the partial sequence of the alligator are presented in Fig. 4. Different amino acid regions exhibit some identity with avian and mammalian proteins (39). These sequences show that the proteins comprise three main domains: two external domains rich in glycine-sequences and with variable identity with mammalian high glycine-tyrosine proteins (39), and a central region rich in proline with good identity to avian scale and feather keratins (Figs 3c, 4 and 5a). The general relationships among these domains and the evolution of these proteins will be discussed later. ...
The structure of reptilian hard (beta)-keratins, their nucleotide and amino acid sequence, and the organization of their genes are presented. These 13-19 kDa proteins are basic, rich in glycine, proline and serine, and different from cytokeratins. Their mRNAs are expressed in beta-cells. The central part of beta-keratins (this region has been previously termed 'core-box' and is peculiar of all sauropsid proteins) is composed of two beta-folded regions and shows a high identity with avian beta-keratins. This central part present in all beta-keratins, including feather keratins, is the site of polymerization to build the framework of beta-keratin filaments. Beta-keratins appear cytokeratin-associated proteins. Their central region might have originated in an ancestral glycine-rich protein present in stem reptiles from which beta-keratins evolved and diversified into reptiles and birds. Stem reptiles of the Carboniferous period might have possessed glycine-rich proteins derived from exons/domains corresponding to the variable, glycine-rich region of cytokeratins. Beta-keratins might have derived from a gene coding for small glycine-rich keratin-associated proteins. The glycine-rich regions evolved differently in the lineage leading to modern reptiles and birds versus that leading to mammals. In the reptilian lineage some amino acid regions produced by point mutations and amino acid changes might have given rise to originate the central beta-pleated region. The latter allowed the formation of filamentous proteins (beta-keratins) associated with intermediate filament keratins and replaced them in beta-keratin cells. In the mammalian lineage no beta-pleated region was generated in their matrix proteins, the glycine-rich keratin-associated proteins. The latter evolved as glycine-tyrosine-rich, sulphur-rich, and ultra-sulphur-rich proteins that are used for building hairs, horns and nails.
... The pioneering findings of Alibardi and co-workers using AE1, AE2, and AE3 antibodies reacting with the epitopes of the mammalian acidic (50-58 kDa) and basic (58-66 kDa) alpha-keratins and some keratins of high molecular weight (66-67 kDa) for the immunolocalization of alpha-keratins in the epidermis of many reptilian species were a milestone for researches of reptilian integument (Alibardi 2004a(Alibardi , 2005Alibardi and Sawyer 2002;Alibardi and Toni 2005;Alibardi et al. 2006Toni and Alibardi 2007a, c). The results of these findings are very important for comparative studies concerning the location and functions of alpha-keratins in the epidermis of reptiles and other amniotic and anamniotic species. ...
... The LH1 in the Oberhäutchen and beta layers at the period of epidermis differentiation in the grass snake perhaps suggests that the layers contain K1/K10-like cytokeratins which create the specific scaffolding for the latest beta-keratin deposition. This is possible because it is known that K1/K10 cytokeratins modify their original role as cytoskeletal components and become important elements for the deposition of other proteins designed to form the intercellular corneous mass and the peripheral cell corneous envelope (Alibardi 2006;Bragulla and Homberger 2009). A similar situation has been observed in other reptilian and avian species (Carver and Sawyer 1987;Shames et al. 1989;Alibardi 2000;Alibardi and Toni 2008). ...
The monoclonal anti-cytokeratin 1/10 (LH1) antibody recognizing K1/K10 keratin epitopes that characterizes a keratinized epidermis of mammals cross-reacts with the beta and Oberhäutchen layers covering the scales and gastrosteges of grass snake embryos during the final period of epidermis differentiation. The immunolocalization of the anti-cytokeratin 1/10 (LH1) antibody appears in the beta layer of the epidermis, covering the outer surface of the gastrosteges at the beginning of developmental stage XI, and in the beta layer of the epidermis, covering the outer surface of the scales at the end of developmental stage XI. This antibody cross-reacts with the Oberhäutchen layers in the epidermis covering the outer surface of both scales and gastrosteges at developmental stages XI and XII just before its fusion with the beta layers. After fusion of the Oberhäutchen and beta layers, LH1 immunolabeling is weaker than before. This might suggest that alpha-keratins in these layers of the epidermis are masked by beta-keratins, modified, or degraded. The anti-cytokeratin 1/10 (LH1) antibody stains the Oberhäutchen layer in the epidermis covering the inner surface of the gastrosteges and the hinge regions between gastrosteges at the end of developmental stage XI. However, the Oberhäutchen of the epidermis covering the inner surfaces of the scales and the hinge regions between scales does not show cytokeratin 1/10 (LH1) immunolabeling until hatching. This cross-reactivity suggests that the beta and Oberhäutchen layers probably contain some alpha-keratins that react with the LH1 antibody. It is possible that these alpha-keratins create specific scaffolding for the latest beta-keratin deposition. It is also possible that the LH1 antibody cross-reacts with other epidermal proteins such as filament-associated proteins, i.e., filaggrin-like. The anti-cytokeratin 1/10 (LH1) antibody does not stain the alpha and mesos layers until hatching. We suppose that the differentiation of these layers will begin just after the first postnatal sloughing.
... To date, only two amino acid sequences of lizard beta keratin are known. One is a 13 kDa sulfur-rich protein isolated from the claw of a Varanus varanus (Inglis et al., 1987); the other is a lizard scale beta keratin (Podarcis sicula) for which the amino acid sequence and the predicted secondary structure have been obtained (Dalla Valle et al., 2005;Alibardi et al., 2006). These small proteins (13-16 kDa) form bundles of corneous material that replace most of the cytokeratin bundles in betacells. ...
... These recent studies indicated that some beta-keratins correspond to glycine-proline-rich proteins that associate to alpha-keratin bundles. These basic proteins are responsible for the hardening of reptilian epidermis, and show some amino acid homology with mammalian keratin-associated proteins Gillespie et al., 1982;Inglis et al., 1987;Alibardi et al., 2006). ...
Snake scales contain specialized hard keratins (beta-keratins) and alpha- or cyto-keratins in their epidermis. The number, isoelectric point, and the evolution of these proteins in snakes and their similarity with those of other vertebrates are not known. In the present study, alpha- and beta-keratins of snake molts and of the whole epidermis have been studied by using two-dimensional electrophoresis and immunocytochemistry. Specific keratins in snake epidermis have been identified by using antibodies that recognize acidic and basic cytokeratins and avian or lizard scale beta-keratin. Alpha keratins of 40-70 kDa and isoelectric point (pI) at 4.5-7.0 are present in molts. The study suggests that cytokeratins in snakes are acidic or neutral, in contrast to mammals and birds where basic keratins are also present. Beta keratins of 10-15 kDa and a pI of 6.5-8.5 are found in molts. Some beta-keratins appear as basic proteins (pI 8.2) comparable to those present in the epidermis of other reptiles. Some basic "beta-keratins" associate with cytokeratins as matrix proteins and replace cytokeratins forming the corneous material of the mature beta-layer of snake scales, as in other reptiles. The study also suggests that more forms of beta-keratins (more than three different types) are present in the epidermis of snakes.
... The putative encoded proteins have high identity in gecko Tm (90% between Ge-gprp-1/2 and Gegprp-3), whereas the two isoforms found in gecko Ht show lower identity (64%), also due to the different lengths between them (Figs. 5, 6, 12). Whereas in the two species of gecko the identity is 56%, between lizard (Dalla Valle et al., 2005;Alibardi et al., 2006) and gecko Tm the identity decrease to 45%, and to 36% between lizard and gecko Ht. ...
... Despite that -keratins are phylogenetically more recent than ␣-keratins of intermediate filaments, their evolution in reptilian epidermis remains unknown (Alibardi et al., 2006). The homology of the central region of gecko (and P. sicula) proteins with scale-feather keratins is interesting, as also the latter protein is polymerized into long cables in elongating barb and barbule cells during feather development (Gregg and Rogers, 1986;Brush, 1993;Fraser and Parry, 1996). ...
The beta-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of beta-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3'- and 5'-rapid amplification of cDNA ends analyses five specific gecko beta-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine-rich proteins of 16.8-18 kDa containing 169-191 amino acids, especially 17.8-23% glycine, 8.4-14.8% proline, 14.2-18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (beta-pleated fold) likely responsible for the formation of beta-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher beta-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and beta-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian beta-keratins.
... Immunocytochemical, proteomic, and molecular biology studies conducted in our laboratory in the last 3 years have collected important cell and molecular information on the process of cornification in reptilian epidermis. 3,[4][5][6][7][8][9][10][11][12] The superficial corneous layer of reptilian scales is lost by a periodic and frequent shedding (5-20 moltings per year in squamates), by an occasional shedding (one to five times per year in the tuatara or one-two times in some turtles), or by a gradual superficial wearing (most chelonians and crocodilians). 10,[13][14][15][16] In lizards and snakes, the epidermis undergoes a cyclical process of resting phase and proliferating (renewal) phase that ends with a molt. ...
... [13][14][15] Alpha-keratins in lizards, snakes, tuatara (Sphenodon punctatus), chelonians, and crocodilians comprise 5-8 low molecular components (40-65 kDa) of acidic type and 5-6 types of neutral-basic type of higher molecular weight (45-68 kDa). 8,[10][11][12][29][30][31][32][33][34] Although the general characteristics and components of alpha-keratins in different reptilian groups have been identified, no molecular data on their sequences and genes are presently available. ...
Beta-keratins form the hard corneous material of reptilian scales. In the present review, the distribution and molecular characteristics of beta-keratins in reptiles are presented. In lepidosaurians immunoreactive, protein bands at 12-18 kDa are generally present with less frequent proteins at higher molecular weight. In chelonians, bands at 13-18 and 22-24 kDa are detected. In crocodilians, bands at 14-20 kDa and weaker bands at 30-32 kDa are seen. Protein bands above 25 kDa are probably polymerized beta-keratins or aggregates. Two-dimensional gel electrophoresis shows that beta-keratins are mainly basic and that acidic-neutral keratins may derive from post-translational modifications. Beta-keratins comprise glycine-proline-rich and cystein-proline-rich proteins of 13-19 kDa. Beta-keratin genes may or may not contain introns and are present in multiple copies with a linear organization as in avian beta-keratin genes. Despite amino acid differences toward N- and C-terminals all beta-keratins share high homology in their central, beta-folded region of 20 amino acids, indicated as core-box. This region is implicated in the formation of beta-keratin filaments of scales, claws, and feathers. The homology of the core-box suggests that these proteins evolved from a progenitor sequence present in the stem of reptiles. Beta-keratins have diversified in their amino acid sequences producing secondary (and tertiary) conformations that suited them for their mechanical role in scales. In birds, a small beta-keratin has allowed the formation of feathers. It is suggested that beta-keratins represent the reptilian counterpart of keratin associated or matrix proteins present in mammalian hairs, claws, and horns.
... The copyright holder for this preprint (which this version posted June 5, 2023. ; β-keratins is largely unknown (Gregg and Rogers 1986;Alibardi et al. 2006). Intriguingly, we observed a tendency to aggregate in GgUox which is dependent on the redox state and mediated by disulfide bond formation (see Fig 6). ...
Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property which we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with β-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.
... Additionally, a broad and intense band between 10 and 12 kDa can be seen. Since β-keratins and α-keratins have molecular masses of 10-22 kDa and 40-68 kDa, respectively [67], it can be said that the extracted keratin contains both types of keratin. The existence of high molecular weights in the extracted keratin indicates that sodium sulfide does not damage protein backbones as observed in previous studies [68,69]. ...
Keratin has attracted substantial interest in the study and development of biomaterials. In this research, the specific extraction method involved the reduction of keratin from chicken feathers using sodium sulfide, and its molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The result showed the presence of β-keratins with lower molecular weight (10–22 kDa) and α-keratins (48–63 kDa). Crosslinked keratin sponges were prepared by glutaraldehyde and using the freeze-drying technique. The response surface method (RSM) based on the central composite rotatable design (CCRD) was used to understand the effects of keratin concentration (2.5 to 10% (w/v)) and glutaraldehyde concentration (0.25 to 1% (w/v)) and their interactions on the ability of sponges to clot whole blood. The optimal keratin sponge was subjected to the ATR-FTIR technique to check the development of the cross-linked polymer network. The SEM images showed regular and interconnected structures, and the pore size was in the range of 100–150 μm. Sponge has a multimodal pore structure with mesopores measuring 5.84 nm (BET surface area: 13.54 m²/g) and 89% of macropores, according to measurements made using nitrogen adsorption porosimetry and volume displacement techniques, respectively. The MTT experiment with human foreskin fibroblasts (HFF) cells showed that the crosslinked keratin sponge had good cell viability. The keratin sponge had high water absorption (91%). The result of whole blood clotting test indicated that keratin plays a significant role in blood coagulation. These sponges with high liquid absorption and blood clotting ability are suitable for hemostatic applications.
Graphic Abstract
... The copyright holder for this preprint (which this version posted June 5, 2023. ; β-keratins is largely unknown (Gregg and Rogers 1986;Alibardi et al. 2006). Intriguingly, we observed a tendency to aggregate in GgUox which is dependent on the redox state and mediated by disulfide bond formation (see Fig 6). ...
Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property which we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H 2 O 2 -oxidative conditions. Cysteine enrichment was concurrent with transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with β-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.
... The latter may strongly bias the differential diagnostic approach and the differentiation of SCCs from BCCs as illustrated by the initial misdiagnosis of eight SCCs that were re-classified as five keratotic BCCs, two infiltrative BCCs, and one solid BCC in the present study. As normal reptile skin typically contains higher amounts of keratin due to the presence of alpha and beta keratin epidermal layers in comparison to humans and other mammals [41], especially keratotic BCCs can be easily misdiagnosed as conventional SCCs or BSCCs because of the abundant presence of keratin pearls [12]. For this reason, it was fundamental to classify keratotic BCCs as a distinct histological variant from solid BCCs in the present study. ...
In the present study, the histological characteristics of squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) obtained from 22 squamate and 13 chelonian species were retrospectively evaluated. While the examined tissues were originally diagnosed as 28 SCCs and 7 BCCs based on histological evaluation by a specialty diagnostic service, eight SCCs could be re-classified as BCCs and three SCCs proved to be non-neoplastic lesions. In addition, all SCCs and BCCs were classified into distinct histological variants. The SCCs could be categorized as one SCC in situ, three moderately differentiated SCCs, seven well-differentiated SCCs, and six keratoacanthomas. BCCs were classified as five solid BCCs, four infiltrating BCCs, five keratotic BCCs, and one basosquamous cell carcinoma. In addition, the present study reports the occurrence of BCCs in seven reptile species for the first time. In contrast to what has been documented in humans, IHC staining with the commercially available epithelial membrane antigen and epithelial antigen clone Ber-EP4 does not allow differentiation of SCCs from BCCs in reptiles, while cyclooxygenase-2 and E-cadherin staining seem to have discriminating potential. Although the gross pathological features of the examined SCCs and BCCs were highly similar, each tumor could be unequivocally assigned to a distinct histological variant according to the observed histological characteristics. Based on the results of this study, a histopathological classification for SCCs and BCCs is proposed, allowing accurate identification and differentiation of SCCs and BCCs and their histological variants in the examined reptile species. Presumably, BCCs are severely underdiagnosed in squamates and chelonians.
... " Wu and Irwin [12]. However, Alibardi et al. [13] had already, on the basis of sequence similarities, pointed to possible evolutionary relationships between keratins of the lizard, the lamprey and even the lancelet and the keratin-associated proteins of the goat, mouse and humans. The present paper is an attempt to dig deeper into this issue, taking into account the many recent additions to the genomic databases. ...
BLAST searches against the human genome showed that of the 93 keratin-associated proteins (KRTAPs) of Homo sapiens, 53 can be linked by sequence similarity to an H. sapiens metallothionein and 16 others can be linked similarly to occludin, while the remaining KRTAPs can themselves be linked to one or other of those 69 directly-linked proteins. The metallothionein-linked KRTAPs comprise the high-sulphur and ultrahigh-sulphur KRTAPs and are larger than the occludin-linked set, which includes the tyrosine- and glycine-containing KRTAPs. KRTAPs linked to metallothionein appeared in increasing numbers as evolution advanced from the deuterostomia, where KRTAP-like proteins with strong sequence similarity to their mammalian congeners were found in a sea anemone and a starfish. Those linked to occludins arose only with the later-evolved mollusca, where a KRTAP homologous with its mammalian congener was found in snails. The presence of antecedents of the mammalian KRTAPs in a starfish, a sea anemone, snails, fish, amphibia, reptiles and birds, all of them animals that lack hair, suggests that some KRTAPs may have a physiological role beyond that of determining the characteristics of hair fibres. We suggest that homologues of these KRTAPs found in non-hairy animals were co-opted by placodes, formed by the ectodysplasin pathway, to produce the first hair-producing cells, the trichocytes of the hair follicles.
... Wu and Irwin (2018) 12 . However, Alibardi etal 2006 13 had already, on the basis of sequence similarities, pointed to possible evolutionary relationships between keratins of the lizard, the lamprey and even the lancelet and the keratin-associated proteins of the goat, mouse and humans. The present paper is an attempt to dig deeper into this issue, taking into account the many recent additions to the genomic databases. ...
BLAST searches against the human genome showed that of the 93 keratin-associated proteins (KRTAPs) of Homo sapiens , 53 can be linked by sequence similarity to an H. sapiens metallothionein and 16 others can be linked similarly to occludin, while the remaining KRTAPs can themselves be linked to one or other of those 69 directly-linked proteins. The metallothionein-linked KRTAPs comprise the high-sulphur and ultrahigh-sulphur KRTAPs and are larger than the occludin-linked set, which includes the tyrosine- and glycine-containing KRTAPs. KRTAPs linked to metallothionein appeared in increasing numbers as evolution advanced from the deuterostomia, where KRTAP-like proteins with strong sequence similarity to their mammalian congeners were found in a sea anemone and a starfish. Those linked to occludins arose only with the later-evolved mollusca, where a KRTAP homologous with its mammalian congener was found in snails. The presence of antecedents of the mammalian KRTAPs in a starfish, a sea anemone, snails, fish, amphibia, reptiles and birds, all of them animals that lack hair, suggests that some KRTAPs may have a physiological role beyond that of determining the characteristics of hair fibres. We suggest that homologues of these KRTAPs found in non-hairy animals were co-opted by placodes, formed by the ectodysplasin pathway, to produce the first hair-producing cells, the trichocytes of the hair follicles.
... It is also instructive to integrate these morphological studies with recent molecular studies on the development of foot scales in modern birds. Earlier developmental and molecular work on avian feathers and scales showed that they are homologous to that of reptilian scales [43][44][45] . It is interesting to note that, histological and molecular investigations on the developmental series of crocodiles and snakes, as well as wild-type lizards and EDA (ectodysplasin A) de cient scaleless mutant lizards reveal that most integumentary structures in amniotes are homologous, which evolved with modi cations from a shared common ancestor characterized by an anatomical placode and a set of signaling molecules 8 . ...
Most modern birds have scales covering the foot, while our knowledge of early avian scales is limited, mainly due to the scarcity of fossil record. Here we characterize the morphological details of two types of scales preserved in IVPP V15077, a referred specimen of the Early Cretaceous bird Gansus . The scutellate and interstitial scales, which, in combination with previous discovery of scutate and reticulate scales in other Early Cretaceous birds, indicates that all four types present in modern birds have appeared in the Early Cretaceous. A phylogenetic context of fossilized scales suggests that the evolution of reticulate scales is conservative while that of the other types is more variable. It is consistent with the molecular hypothesis of the scales in modern birds and reptiles that most integumentary structures in amniotes are homologous with modified signaling modules to form various integumentary phenotypes, among which the reticulate scales may use the conserved signaling pathway.
... Electron microscopy investigations of the cross-sections of the hard keratins constituting the epidermal appendages (feathers, claws, scales) of the sauropsids have shown that these tissues have a filament-matrix texture [6][7][8][9][10][11][12]. This is dominated by filaments about 3.4 nm in diameter that provide much of the tensile strength of the tissue (Figure 2). ...
The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7–10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.
... Several genomic studies indicate that feather β-keratin genes were probably present in the genome and expressed in the epidermis of scaled archosaurians and lepidosaurs before the emergence of feathers (31)(32)(33)(34). However, the formation of feather placodes and the origin of the axial rachis and hierarchical branching of barbs and barbules, as well as different feather types with different functions, required increasing specialization of the feather β-keratin genes (15). ...
During the dinosaur–bird transition, feathers of bird ancestors must have been molecularly modified to become biomechanically suitable for flight. We report molecular moieties in fossil feathers that shed light on that transition. Pennaceous feathers attached to the right forelimb of the Jurassic dinosaur Anchiornis were composed of both feather β-keratins and α-keratins, but were dominated by α-keratins, unlike mature feathers of extant birds, which are dominated by β-keratins. Data suggest that the pennaceous feathers of Anchiornis had some, but not all, of the ultrastructural and molecular characteristics of extant feathers, and may not yet have attained molecular modifications required for powered flight.
Dinosaur fossils possessing integumentary appendages of various morphologies, interpreted as feathers, have greatly enhanced our understanding of the evolutionary link between birds and dinosaurs, as well as the origins of feathers and avian flight. In extant birds, the unique expression and amino acid composition of proteins in mature feathers have been shown to determine their biomechanical properties, such as hardness, resilience, and plasticity. Here, we provide molecular and ultrastructural evidence that the pennaceous feathers of the Jurassic nonavian dinosaur Anchiornis were composed of both feather β-keratins and α-keratins. This is significant, because mature feathers in extant birds are dominated by β-keratins, particularly in the barbs and barbules forming the vane. We confirm here that feathers were modified at both molecular and morphological levels to obtain the biomechanical properties for flight during the dinosaur–bird transition, and we show that the patterns and timing of adaptive change at the molecular level can be directly addressed in exceptionally preserved fossils in deep time.
... In the case of β-keratin, there are no different types of proteins (Fraser et al. 1972), as the filaments and matrix are incorporated into one single protein (Fraser and Parry 2011). The molecular mass of α-keratin ranged from 40 kDa to 68 kDa that is to a large extent bigger than that of β-keratin that is 10-22 kDa (Alibardi et al. 2006). ...
Keratins are insoluble, fibrous, and structural proteins that are present in the epidermis and its appendages and these include feather, hair, wool, nail, hoof, and horns. Keratins adhere epidermal cells to one another and provide protection on the skin. They are structurally stabilized by their tightly packed peptide chains and the existence of several cross-linkages by disulphide bonds, hydrogen bonding, and hydrophobic interactions. Keratin-containing materials are generated abundantly as by-products of agro-industrial processing and constitute nuisance in the environment as a result of their recalcitrance to degradation by regular proteolytic enzymes like pepsin, trypsin, and papain. The traditional physical and chemical techniques for their treatment are expensive, energy consuming, can damage some essential amino acids, and non-environmentally benign. However, degradation by a variety of microorganisms had proven to be a viable alternative means of keratin treatment. A vast variety of bacteria, fungi, and actinomycetes have been recognized as keratin degraders. They degrade keratins mainly with their keratinases, which sometimes act synergistically with other enzymes like disulfide reductases and cysteine dioxygenase for effective degradation of keratins. The microbial keratinases hydrolyze keratins into soluble proteins, peptides, and amino acids. They are utility enzymes with very diverse biotechnological applications. Biodegradation of keratin-rich wastes by microorganisms is therefore an efficient, cheap, and eco-friendly method of waste management and production of products of high biotechnological value. The present review examines the trends in the role of microorganisms for the biotechnological treatment of keratin-rich wastes.
... keratins suitable for biomedical applications. Two versions of this method were originally described in references [39] and [40], the second one being reported as effective for solubilizing β-keratins [41]. An efficient alternative is presented in reference [42], and reference [43] reports a method for γ-keratins isolation and purification. ...
Keratin is a ubiquitous presence in vertebrata subphylum, as intracellular intermediate filaments, and is visible and abundant as morphological structures at the level of integuments and their products. The keratin sources and resources are exploitable for usual textile goods and chemical auxiliaries production, but there are of obvious interest for advanced applications, like biomaterials and tissue engineering. The condition to include keratin species in such applications is to extract them in a quasi-native state, starting from pristine sources. The present paper briefly discuss the properties which sustain the use of keratins in biomedical applications, together with some principles of solubilizing keratin sources in a protective manner, so that new material characteristics to result by specific processing.
... Hydrogen peroxide, provided in the ECL-solutions, is the substrate of the peroxidase and thus oxidized so that the protons released during the reaction lead to the formation of luminal radicals, showing chemiluminescence when decaying and returning to the ground state [12]. 20 mg of moulted skin of S. scincus were incubated at room temperature, protected from light, overnight in a lysis buffer consisting of (after [13]): 267 µl 9M Urea, 20 µl 1.5M Tris/HCl pH 7.6, 5.2 µl PMSF, 2.1 µl β-mercaptoethanol and 5.7 µl dH 2 O. Afterwards, samples were purified from skin residues and the protein concentration was measured photometrically at 578 nm by a Bradford assay [14] with Bradford reagent (Roti®Quant, Roth, Karlsruhe, Germany), whereby the proteins for reference concentration measurement (BSA, Sigma-Aldrich) were dissolved in the lysis buffer consisting of the same chemical compounds. After preparation with laemmli buffer containing 2.5% 2-mercaptoethanol [15], protein samples were separated by a 15% SDS-PAGE containing 5 µg of lysed protein each lane. ...
The sandfish (Scincidae: Scincus scincus) is a lizard capable of moving through desert sand in a swimming-like fashion. The epidermis of this lizard shows a high resistance against abrasion together with a low friction to sand as an adaption to a subterranean life below the desert's surface, outperforming even steel. The low friction is mainly caused by chemical composition of the scales, which consist of glycosylated β-keratins. In this study, the friction, the micro-structure, the glycosylation of the β-keratin proteins and β-keratin coding DNA of the sandfish in comparison to other reptilian species was investigated, mainly with the closely related Berber skink (Scincidae: Eumeces schneideri) and another sand swimming species, the not closer related Shovel-snouted lizard (Lacertidae: Meroles anchietae). Glycosylated β-keratins of the sandfish, visualized with different lectins resulted in O-linked glycans through PNA employed as carbohydrate marker. Furthermore, the glycosylation of β-keratins in various squamatean species was investigated and all species tested were found positive; however, it seems like both sand swimming species examined have a much stronger glycosylation of their β-keratins. In order to prove this finding through a genetic foundation, DNA of a β-keratin coding gene of the sandfish was sequenced and compared with a homologue gene of Eumeces schneideri. By comparison of the protein sequence, a higher abundance of O-glycosylation sites was found in the sandfish (enabled through the amino acids serine and threonine), giving molecular support for a higher glycosylation of the β-keratins in this species.
... Other genes coding for small high glycine-serine proteins (HGSs) produced multiple forms of KAPs, present in the interkeratin or matrix material of hairs, claws, feathers, horns, etc. Matrix proteins could be formed in the ancestors of reptiles since the Upper Carboniferous, more than 300 million years ago. These small proteins (the HGSs) evolved differently in amniotes, and produced glycine-rich and sulphur-rich 'beta-keratins', in reptiles and birds, today identified as sauropsid KAPs (sKAPs, see Alibardi et al. 2006bToni et al. 2007). In theropsid reptiles (synapsids and the derived therapsids and extant mammals), the ancestral proteins (HGSs) evolved into glycine-tyrosine-rich and sulphur-rich mKAPs (Fig. 11). ...
Abstract Alibardi, L. 2009. Review: Embryonic keratinization in vertebrates in relation to land colonization. —Acta Zoologica (Stockolm) 90: 1–17 The embryogenesis and cytology of the epidermis in different vertebrates is variable in relation to the formation of a stratum corneum of different complexity. The latter process was essential for land colonization during vertebrate evolution and produced an efficient barrier in amniotes. Keratinocytes are made of cross-linked keratins associated with specific proteins and lipids that are produced at advanced stages of embryogenesis when the epidermis becomes stratified. In these stages the epidermis changes from an aquatic to a terrestrial type, preadapted in preparation for the impact with the dry terrestrial environment that occurs at hatching or parturition. The epidermal barrier against water-loss, mechanical and chemical stress, and microbe penetration is completely formed shortly before birth. Beneath the outer periderm, variably stratified embryonic layers containing glycine-rich alpha-keratins are formed in preparation for adult life. The following layers of the epidermis produce proteins for the formation of the cornified cell membrane and of the cornified material present in keratinocytes of the adult epidermis in reptiles, birds and mammals. The general features of the process of soft cornification in the embryonic epidermis of vertebrates are presented. Cornification in developing scales in reptiles, avian feathers and mammalian hairs is mainly related to the evolution of keratin-associated proteins. The latter proteins form the resistant matrix of hard skin derivatives such as claws, beaks, nails and horns.
... HRPs have also been described in lizard epidermis (Alibardi et al., 2004). Furthermore, studies of lizard claw keratin-revealed functional domains or segments whose amino acid sequences resembled either the a-keratins of mammals or the b-keratins of feathers (Inglis et al., '87;Fraser and Parry, '96;Alibardi et al., 2006a), supporting the view that the b-keratins most likely evolved from the older a-keratins found in all vertebrates '86;Klinge et al.,'87). ...
Feathers of today's birds are constructed of beta (β)-keratins, structural proteins of the epidermis that are found solely in reptiles and birds. Discoveries of "feathered dinosaurs" continue to stimulate interest in the evolutionary origin of feathers, but few studies have attempted to link the molecular evolution of their major structural proteins (β-keratins) to the appearance of feathers in the fossil record. Using molecular dating methods, we show that before the appearance of Anchiornis (∼155 Million years ago (Ma)) the basal β-keratins of birds began diverging from their archosaurian ancestor ∼216 Ma. However, the subfamily of feather β-keratins, as found in living birds, did not begin diverging until ∼143 Ma. Thus, the pennaceous feathers on Anchiornis, while being constructed of avian β-keratins, most likely did not contain the feather β-keratins found in the feathers of modern birds. Our results demonstrate that the evolutionary origin of feathers does not coincide with the molecular evolution of the feather β-keratins found in modern birds. More likely, during the Late Jurassic, the epidermal structures that appeared on organisms in the lineage leading to birds, including early forms of feathers, were constructed of avian β-keratins other than those found in the feathers of modern birds. Recent biophysical studies of the β-keratins in feathers support the view that the appearance of the subfamily of feather β-keratins altered the biophysical nature of the feather establishing its role in powered flight.
... The use here of the ''high'' and ''low'' terminology to classify these proteins has been adopted for comparison of sauropsid KAPs with mammalian KAPs, the latter generally classified as high glycine tyrosine I and II proteins (HTP I and II), high sulfur proteins (HSP), and ultra-high sulfur proteins (UHSP; see Powell and Rogers,'94). In previous papers on the characterization of b-keratins, the terms cysteine-rich and glycine-rich proteins were also utilized (Dalla Valle et al., 2006Valle et al., , b, 2009a. ...
Using bioinformatic methods we have detected the genes of 40 keratin-associated beta-proteins (KAbetaPs) (beta-keratins) from the first available draft genome sequence of a reptile, the lizard Anolis carolinensis (Broad Institute, Boston). All genes are clustered in a single but not yet identified chromosomal locus, and contain a single intron of variable length. 5'-RACE and RT-PCR analyses using RNA from different epidermal regions show tissue-specific expression of different transcripts. These results were confirmed from the analysis of the A. carolinensis EST libraries (Broad Institute). Most deduced proteins are 12-16 kDa with a pI of 7.5-8.5. Two genes encoding putative proteins of 40 and 45 kDa are also present. Despite variability in amino acid sequences, four main subfamilies can be described. The largest subfamily includes proteins high in glycine, a small subfamily contains proteins high in cysteine, a third large subfamily contains proteins high in cysteine and glycine, and the fourth, smallest subfamily comprises proteins low in cysteine and glycine. An inner region of high amino acid identity is the most constant characteristic of these proteins and maps to a region with two to three close beta-folds in the proteins. This beta-fold region is responsible for the formation of filaments of the corneous material in all types of scales in this species. Phylogenetic analysis shows that A. carolinensis KAbetaPs are more similar to those of other lepidosaurians (snake, lizard, and gecko lizard) than to those of archosaurians (chick and crocodile) and turtles.
... Amino acid sequences of b-keratins and their predicted secondary structures are now available for some lepidosaurian reptiles, such as lizards and snakes (Dalla Valle et al., 2005;Alibardi et al., 2006a,b;Dalla Valle et al., 2007a,b). These proteins form filaments of b-keratins and have been recently named ''sauropsid keratin-associated proteins,'' because they represent the reptilian-avian counterpart of ''mammalian keratin-associated proteins'' (Alibardi et al., 2006a(Alibardi et al., ,b, 2007Toni et al., 2007a). In lizards and snakes so far studied, these proteins contain high amounts of glycine, serine, and proline, the latter amino acid frequently localizing to a central region designated the ''core box.'' ...
Nucleotide and deduced amino acid sequences of three beta-keratins of Nile crocodile scales are presented. Using 5'- and 3'-RACE analysis, two cDNA sequences of 1 kb (Cr-gptrp-1) and 1.5 kb (Cr-gptrp-2) were determined, corresponding to 17.4 and 19.3 kDa proteins, respectively, and a pI of 8.0. In genomic DNA amplifications, we determined that the 5'-UTR of Cr-gptrp-2 contains an intron of 621 nucleotides. In addition, we isolated a third gene (Cr-gptrp-3) in genomic DNA amplifications that exhibits seven amino acid differences with Cr-gptrp-2. Genomic organization of the sequenced crocodilian beta-keratin genes is similar to avian beta-keratin genes. Deduced proteins are rich in glycine, proline, serine, and tyrosine, and contain cysteines toward the N- and C-terminal regions, likely for the formation of disulfide bonds. Prediction of the secondary structure suggests that the central core box of 20 amino acids contains two beta-strands and has 75-90% identity with chick beta-keratins. Toward the C-terminus, numerous glycine-glycine-tyrosine and glycine-glycine-leucine repeats are present, which may contribute to making crocodile scales hard. In situ hybridization shows expression of beta-keratin genes in differentiating beta-cells of epidermal transitional layers. Phylogenetic analysis of the available archosaurian and lepidosaurian beta-keratins suggests that feather keratins diversified early from nonfeather keratins, deep in archosaur evolution. However, only the complete knowledge of all crocodilian beta-keratins will confirm whether feather keratins have an origin independent of those in bird scales, which preceded the split between birds and crocodiles.
Protein-based biomaterials have emerged as a promising alternative because of their inherent cell-to-cell interaction, structural support, and cellular communications. Over the last century, advances in the extraction, purification, and characterization of keratin proteins from wool, feathers, horns, and other animal sources have created a keratin-based biomaterials platform. Keratins, like many other naturally generated macromolecules, have biological activity and biocompatibility built-in. Furthermore, isolated keratins can self-assemble into structures that control cellular identification and behaviour. As a result, keratin biomaterials with applications in wound healing, drug delivery, tissue engineering, trauma, and medical devices have been developed due to these properties. This review examines the function of keratin in the human body in-depth, focusing on the history of keratin research and a current evaluation of emerging approaches in biomedical fields like tissue engineering, medical science, regenerative medicine, and drug delivery.
Keratins, as a group of insoluble and filament-forming proteins, mainly exist in certain epithelial cells of vertebrates. Keratinous materials are made up of cells filled with keratins, while they are the toughest biological materials such as the human hair, wool and horns of mammals and feathers, claws, and beaks of birds and reptiles which usually used for protection, defense, hunting and as armor. They generally exhibit a sophisticated hierarchical structure ranging from nanoscale to centimeter-scale: polypeptide chain structures, intermediated filaments/matrix structures, and lamellar structures. Therefore, more and more attention has been paid to the investigation of the relationship between structure and properties of keratins, and a series of biomimetic materials based on keratin came into being. In this chapter, we mainly introduce the hierarchical structure, the secondary structure, and the molecular structure of keratins, including α- and β-keratin, to promote the development of novel keratin-based biomimetic materials designs.
The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent β-keratin proteins (also referred to as the corneous β-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the β-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the β-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).
Keratin is a global class of biological material, which represents a group of cysteine-rich filament-forming proteins. They serve as a shielding layer for the epidermal appendages like nails, claws, beak, hair, wool, horns, and feathers. These proteins are further subdivided into two different class based on their secondary structure: α-keratin and β-keratin. Keratin is insoluble in hot or cold water; this unique property helps to prevent their digestion by proteolytic enzymes. Additionally, their complex hierarchical-like filament-matrix structure at nanoscale and the polypeptide chains create a robust wall for protection against heat stress, pathogen invasions (particularly through skin), mechanical damage, etc. In this review, we are trying to attempt a linear focus in the direction of structure, function, extraction of keratin, and its industrial applications.
The structures of avian and reptilian epidermal appendages, such as feathers, claws and scales, have been modelled using X-ray diffraction and electron microscopy data, combined with sequence analyses. In most cases, a family of closely related molecules makes up the bulk of the appendage, and each of these molecules contains a central β-rich 34-residue segment, which has been identified as the principal component of the framework of the 3.4 nm diameter filaments. The N- and C-terminal segments form the matrix component of the filament/matrix complex. The 34-residue β-rich central domains occur in pairs, related by either a parallel dyad or a perpendicular dyad axis, and form a β-sandwich stabilized by apolar interactions. They are also twisted in a right-handed manner. In feather, the filaments are packed into small sheets and it is possible to determine their likely orientation within the sheets from the low-angle X-ray diffraction data. The physical properties of the various epidermal appendages can be related to the amino acid sequence and composition of defined molecular segments characteristic of the chains concerned.
Significance
We report fossil evidence of feather structural protein (beta-keratin) from a 130-My-old basal bird ( Eoconfuciusornis ) from the famous Early Cretaceous Jehol Biota, which has produced many feathered dinosaurs, early birds, and mammals. Multiple independent molecular analyses of both microbodies and associated matrix recovered from the fossil feathers confirm that these microbodies are indeed melanosomes. We use transmission electron microscopy and immunogold to show localized binding of antibodies raised against feather protein to matrix filaments within these ancient feathers. Our work sheds new light on molecular constituents of tissues preserved in fossils.
Avian and reptilian epidermal appendages such as feathers, claws and scales exhibit a filament-matrix texture. Previous studies have established that both components reside within the same single-chain molecule. In the present study the homology in a wide range of aligned sequences is used to gain insights into the structure and function of the molecular segments associated with the filament and with the matrix. The notion that all molecules contain a β-rich 34-residue segment associated with the framework of the filament is reinforced by the present study. In addition, the residues involved in the polymerization of the molecules to form filaments are identified. In the Archosaurs (birds, crocodiles and turtles), and the Squamates (snakes and lizards) segments rich in glycine and tyrosine can be identified in the C-terminal domain. In Rhynocephalians (tuataras) and Squamates a similar segment is inserted at a specific point in the N-terminal domain. In some Archosaurian appendages (both avian and reptilian) segments rich in charged residues and cysteine are found in the N-terminal domain. The likely effect of these segments will be to soften the tissue without compromising its insolubility. The structure and role of the various molecular segments identified in this study and the way in which they might manifest themselves in terms of the physical properties of the particular epidermal appendage in which they appear are also discussed.
The presence of beta-proteins containing a core-box region in specific regions of reptilian epidermis has been studied by immunological methods. Alpha-keratins are detected by the antibody AK2 that recognizes a sequence toward the C-terminal of acidic alpha-keratins of 48-52kDa. Beta-proteins are recognized by an antibody directed to the core-box region specific for these proteins of 18-37kDa. The AK2 antibody labels with variable intensity alpha-keratin bundles in basal and suprabasal keratinocytes in the epidermis of representative species of reptiles but immunolabeling decreases or disappears in pre-corneous and corneous cells. As opposite, the core-box antibody only labels with variable intensity the dense beta-corneous material formed in pre-corneous and corneous layers of crocodilian and turtle epidermis. In lepidosaurian epidermis the core-box antibody labels the beta-layer while the mesos and alpha-layers are poorly or not labeled. The immunological evidence indicates that beta-proteins are synthesized in the upper spinosus and pre-corneous layers of the epidermis and replace or mask the initial alpha-keratin framework present in keratinocytes as they differentiate into cells of the beta-layer. In the specialized pad lamellae of gecko and anoline lizards charged beta-proteins accumulate in the adhesive setae and may affect the mechanism of adhesion that allows these lizards to walk vertical surfaces. The addition of beta-proteins to the alpha-keratins in upper cell layers of the epidermis recalls the process of cornification of mammalian epidermis where specific keratin-associated proteins (involucrin, loricrin and filaggrin) associate with the keratin framework in terminally differentiating keratinocytes of the stratum corneum.
Morphogenesis of claws in the lizard Lampropholis guichenoti has been studied by light and electron microscopy. Claws originate from a thickening of the epidermis covering the tips of digits under which mesenchymal cells aggregate. Mesenchymal cells are in continuity with perichondrial cells of the last phalange, and are connected to the epidermis through numerous cell bridges that cross an incomplete basement membrane. The dense lamella is completed in non-apical regions of the digit where also collagen fibrils increase. The dorsal side of the developing claw derives from the growth of the outer scale surface of the last scale of the digit. The corneous layer, made of beta-keratin cells, curves downward by the tip of the growing claw. The epidermis of the ventral side of the claw contains keratohyaline-like granules and alpha-keratinocytes like an inner scale surface. The thickness of the horny layer increases in the elongating unguis while a thinner and softer corneous layer remains in the subunguis. These observations show that lizard claws derive from the modification of the last scale or scales of the digit, probably under the influence of the growing terminal phalanx. Some hypotheses on the evolution of claws in reptiles are presented.
Feather keratin has a composite structure with a filament-matrix texture, and transmission electron microscopy studies of thin transverse sections of feather rachis by Rogers and Filshie in the early 1960s showed that the filaments have a strong tendency to form sheets. Potentially this could account for the unusual X-ray diffraction pattern noted by Bear and Rugo in the early 1950s, which was interpreted by them as indicating a two-dimensional net structure. Although it is 50years since these major advances were made the possibility of extracting information on the nature of the filament packing from the diffraction pattern has never been explored. The present contribution shows how, when taken together with current information on the nature of β-sheets in feather keratin, certain features of the X-ray diffraction pattern can now be used to determine the likely arrangement of the filaments in the sheet.
Avian hard keratin has a filament-matrix texture in which the filaments contain a helical array of twisted β-sheets and the matrix has unusually high concentrations of cysteine, glycine, and tyrosine. X-ray diffraction studies have established that similar filaments exist in the hard keratins of crocodiles, turtles, tuataras, lizards and snakes. Here, the relationship between amino acid sequence and the filament-matrix texture is explored in a wide variety of avian and reptilian hard keratins. Universally, the molecules contain three distinct domains: a central domain rich in β-favoring residues associated with the filament framework, and N- and C-terminal domains associated with the matrix and with crosslinking via disulfide bonds. A variety of structural probes were employed to identify the β-framework of the filaments and a common pattern 34 residues in length was found in all cases. In addition, detailed analyses of the sequences in the two "matrix" domains revealed profound differences between the Archosaurs (birds, crocodiles and turtles), where the N-terminal domains were very similar, and the Squamates (snakes and lizards) where the N-terminal domains varied widely in length and composition, in some cases exhibiting a subdomain structure, and segments of highly homologous sequence. The C-terminal domains in both branches varied widely in composition but almost all exhibit a subdomain structure characterized by a terminal sequence rich in cysteine and arginine residues. A revised model for the molecular organization in avian and reptilian hard keratins is presented and similarities and differences in the matrix domains are noted.
Adhesive devices of digital pads of gecko lizards are formed by microscopic hair-like structures termed setae that derive from the interaction between the oberhautchen and the clear layer of the epidermis. The two layers form the shedding complex and permit skin shedding in lizards. Setae consist of a resistant but flexible corneous material largely made of keratin-associated beta-proteins (KA beta Ps, formerly called beta-keratins) of 8-22 kDa and of alpha-keratins of 45-60 kDa. In Gekko gecko, 19 sauropsid keratin-associated beta-proteins (sKAbetaPs) and at least two larger alpha-keratins are expressed in the setae. Some sKA beta Ps are rich in cysteine (111-114 amino acids), while others are rich in glycine (169-219 amino acids). In the entire genome of Anolis carolinensis 40 Ka beta Ps are present and participate in the formation of all types of scales, pad lamellae and claws. Nineteen sKA beta Ps comprise cysteine-rich 9.2-14.4 kDa proteins of 89-142 amino acids, and 19 are glycine-rich 16.5-22.0 kDa proteins containing 162-225 amino acids, and only two types of sKA beta Ps are cysteine- and glycine-poor proteins. Genes coding for these proteins contain an intron in the 5'-non-coding region, a typical characteristic of most sauropsid Ka beta Ps. Gecko KA beta Ps show a central amino acid region of high homology and a beta-pleated conformation that is likely responsible for the polymerization of Ka beta Ps into long and resistant filaments. The association of numerous filaments, probably over a framework of alpha-keratins, permits the formation of bundles of corneous material for the elongation of setae, which may be over 100 microm long. The terminals branching off each seta may derive from the organization of the cytoskeleton and from the mechanical separation of keratin bundles located at the terminal apex of setae.
Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages probably derived from the localization of specialized areas of dermal-epidermal interaction in the integument. The horny scales and the other derivatives were formed from large areas of dermal-epidermal interaction. The evolution of these skin appendages was characterized by the production of specific coiled-coil keratins and associated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsids contain a double beta-folded sequence of about 20 amino acids, known as the core-box. The core-box shows 60%-95% sequence identity with known reptilian and avian proteins. The core-box determines the polymerization of these proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequences for these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolution in reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestral glycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrix proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral protein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently in the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high glycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, which evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In therapsids-mammals, mutations of the ancestral proteins formed the high glycine-tyrosine or the high cysteine proteins but no core-box was produced in the matrix proteins of the hard corneous material of mammalian derivatives.
The DNA sequences encoding beta-keratin have been obtained from Marsh Mugger (Crocodylus palustris) and Orinoco Crocodiles (Crocodylus intermedius). Through the deduced amino acid sequence, these proteins are rich in glycine, proline and serine. The central region of the proteins are composed of two beta-folded regions and show a high degree of identity with beta-keratins of aves and squamates. This central part is thought to be the site of polymerization to build the framework of beta-keratin filaments. It is believed that the beta-keratins in reptiles and birds share a common ancestry. Near the C-terminal, these beta-keratins contain a peptide rich in glycine-X and glycine-X-X, and the distinctive feature of the region is some 12-amino acid repeats, which are similar to the 13-amino acid repeats in chick scale keratin but absent from avian feather keratin. From our phylogenetic analysis, the beta-keratins in crocodile have a closer relationship with avian keratins than the other keratins in reptiles.
This study presents, for the first time, sequences of five beta-keratin cDNAs from turtle epidermis obtained by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The deduced amino acid sequences correspond to distinct glycine-proline-serine-tyrosine rich proteins containing 122-174 amino acids. In situ hybridization shows that beta-keratin mRNAs are expressed in cells of the differentiating beta-layers of the shell scutes. Southern blotting analysis reveals that turtle beta-keratins belong to a well-conserved multigene family. This result was confirmed by the amplification and sequencing of 13 genomic fragments corresponding to beta-keratin genes. Like snake, crocodile and avian beta-keratin genes, turtle beta-keratins contain an intron that interrupts the 5'-untranslated region. The length of the intron is variable, ranging from 0.35 to 1.00 kb. One of the sequences obtained from genomic amplifications corresponds to one of the five sequences obtained from cDNA cloning; thus, sequences of a total of 17 turtle beta-keratins were determined in the present study. The predicted molecular weight of the 17 different deduced proteins range from 11.9 to 17.0 kDa with a predicted isoelectric point of 6.8-8.4; therefore, they are neutral to basic proteins. A central region rich in proline and with beta-strand conformation shows high conservation with other reptilian and avian beta-keratins, and it is likely involved in their polymerization. Glycine repeat regions, often containing tyrosine, are localized toward the C-terminus. Phylogenetic analysis shows that turtle beta-keratins are more similar to crocodilian and avian beta-keratins than to those of lizards and snakes.
Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.
Lizard scales are composed of alpha-(cyto-) keratins and beta-keratins. The characterization of the molecular weight and isoelectric point (pI) of alpha- and beta-keratins of lizard epidermis (Podarcis sicula) has been done by using two-dimensional electrophoresis, immunoblotting, and immunocytochemistry. Antibodies against cytokeratins, against a chicken scale beta-keratin or against lizard beta-keratin bands of 15-16kDa, have been used to recognize alpha- and beta-keratins. Acid and basic cytokeratins of 42-67kDa show a pI from 5.0 to 8.9. This indicates the presence of specific keratins for the formation of the stratum corneum. Main protein spots of beta-keratin at 15-17kDa, and pI at 8.5, 8.2, and 6.7, and one spot at 10kDa and pI at 7.3 were recognized. Therefore, beta-keratins are mainly basic proteins, and are used for the formation of the hard corneous layer of the epidermis. Ultrastructural immunocytochemistry confirms that beta-keratin is packed into large and dense bundles of beta-keratin cells of lizard epidermis. The use of a probe against a lizard beta-keratin in situ-hybridization studies confirms that the mRNA for beta-keratins is present in beta-cells and is localized around or even associated with beta-keratin filaments.
Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine-proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine-proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life.
Beta-keratins are responsible for the mechanical resistance of scales in reptiles. In a scaleless crotalus snake (Crotalus atrox), large areas of the skin are completely devoid of scales, and the skin appears delicate and wrinkled. The epidermis of this snake has been assessed for the presence of beta-keratin by immunocytochemistry and immunoblotting using an antibody against chicken scale beta-keratin. This antibody recognizes beta-keratins in normal snake scales with molecular weights of 15-18 kDa and isoelectric points at 6.8, 7.5, 8.3 and 9.4. This indicates that beta-keratins of the stratum corneum are mainly basic proteins, so may interact with cytokeratins of the epidermis, most of which appear acidic (isoelectric points 4.5-5.5). A beta-layer and beta-keratin immunoreactivity are completely absent in moults of the scaleless mutant, and the corneous layer comprises a multi-layered alpha-layer covered by a flat oberhautchen. In conclusion, the present study shows that a lack of beta-keratins is correlated with the loss of scales and mechanical protection in the skin of this mutant snake.
Crocodilian keratinocytes accumulate keratin and form a corneous cell envelope of which the composition is poorly known. The present immunological study characterizes the molecular weight, isoelectric point (pI) and the protein pattern of alpha- and beta-keratins in the epidermis of crocodilians. Some acidic alpha-keratins of 47-68 kDa are present. Cross-reactive bands for loricrin (70, 66, 55 kDa), sciellin (66, 55-57 kDa), and filaggrin-AE2-positive keratins (67, 55 kDa) are detected while caveolin is absent. These proteins may participate in the formation of the cornified cell membranes, especially in hinge regions among scales. Beta-keratins of 17-20 kDa and of prevalent basic pI (7.0-8.4) are also present. Acidic beta-keratins of 10-16 kDa are scarce and may represent altered forms of the original basic proteins. Crocodilian beta-keratins are not recognized by a lizard beta-keratin antibody (A68B), and by a turtle beta-keratin antibody (A685). This result indicates that these antibodies recognize specific epitopes in different reptiles. Conversely, crocodilian beta-keratins cross-react with the Beta-universal antibody indicating they share a specific 20 amino acid epitope with avian beta-keratins. Although crocodilian beta-keratins are larger proteins than those present in birds our results indicate presence of shared epitopes between avian and crocodilian beta-keratins which give good indication for the future determination of the sequence of these proteins.
Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as "feather keratin". This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.
A book edited by J.Joseph Marr and Miklos Müller that sketches a picture of the state of the art of the biochemical research on parasites.
We describe a new formulation for a hydrophilic resin, mostly composed of glycol methacrylate and hydroxypropyl methacrylate and here referred to as bioacryl, that allows the performance of morphological and immunohistochemical investigations at both light and electron microscopic levels. Immunolocalizations performed on bioacryl-embedded tissues are characterized by high specificity with virtually absent background staining. Finally, the new resin yields satisfactory fine-structural preservation, resulting in ultrastructural images of better quality than those obtained with Lowicryl K4M.
The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.
The adaptation to land from amphibians to amniotes was accompanied by drastic changes of the integument, some of which might be reconstructed by studying the formation of the stratum corneum during embryogenesis. As the first amniotes were reptiles, the present review focuses on past and recent information on the evolution of reptilian epidermis and the stratum corneum. We aim to generalize the discussion on the evolution of the skin in amniotes. Corneous cell envelopes were absent in fish, and first appeared in adult amphibian epidermis. Stem reptiles evolved a multilayered stratum corneum based on a programmed cell death, intensified the production of matrix proteins (e.g., HRPs), corneous cell envelope proteins (e.g., loricrine-like, sciellin-like, and transglutaminase), and complex lipids to limit water loss. Other proteins were later produced in association to the soft or hairy epidermis in therapsids (e.g., involucrin, profilaggrin-filaggrin, trichohyalin, trichocytic keratins), or to the hard keratin of hairs, quills, horns, claws (e.g., tyrosine-rich, glycine-rich, sulphur-rich matrix proteins). In sauropsids special proteins associated to hard keratinization in scales (e.g., scale beta-keratins, cytokeratin associated proteins) or feathers (feather beta-keratins and HRPs) were originated. The temporal deposition of beta-keratin in lepidosaurian reptiles originated a vertical stratified epidermis and an intraepidermal shedding layer. The evolutions of the horny layer in Therapsids (mammals) and Saurospids (reptiles and birds) are discussed. The study of the molecules involved in the dermo-epidermal interactions in reptilian skin and the molecular biology of epidermal proteins are among the most urgent future areas of research in the biology of reptilian skin.
During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.
Ten years ago, Hardy (1992) wrote a timely review on the major features of hair follicle development and hair growth which she referred to as a secret life. Many of these secrets are now being revealed. The information discussed in this brief review comprises the structure of the hair and hair follicle, the continuing characterisation of the genes for keratin and keratin associated proteins, the determination of the location of their expression in the different cell layers of the hair follicle, molecular signals which control keratin gene expression and post-translational events in the terminal stages of hair formation.
Reptilian epidermis contains two types of keratin, soft (alpha) and hard (beta). The biosynthesis and molecular weight of beta-keratin during differentiation of lizard epidermis have been studied by autoradiography, immunocytochemistry and immunoblotting. Tritiated proline is mainly incorporated into differentiating and maturing beta-keratin cells with a pattern similar to that observed after immunostaining with a chicken beta-keratin antibody. While the antibody labels a mature form of beta-keratin incorporated in large filaments, the autoradiographic analysis shows that beta-keratin is produced within the first 30 min in ribosomes, and is later packed into large filaments. Also the dermis incorporates high amount of proline for the synthesis of collagen. The skin was separated into epidermis and dermis, which were analyzed separately by protein extraction and electrophoresis. In the epidermal extract proline-labeled proteic bands at 10, 15, 18-20, 42-45, 52-56, 85-90 and 120 kDa appear at 1, 3 and 5 h post-injection. The comparison with the dermal extract shows only the 85-90 and 120 kDa bands, which correspond to collagen. Probably the glycine-rich sequences of collagen present also in beta-keratins are weakly recognized by the beta-1 antibody. Immunoblotting with the beta-keratin antibody identifies proteic bands according to the isolation method. After-saline or urea-thiol extraction bands at 10-15, 18-20, 40, 55 and 62 kDa appear. After extraction and carboxymethylation, weak bands at 10-15, 18-20 and 30-32 kDa are present in some preparations, while in others also bands at 55 and 62 kDa are present. It appears that the lowermost bands at 10-20 kDa are simple beta-keratins, while those at 42-56 kDa are complex or polymeric forms of beta-keratins. The smallest beta-keratins (10-20 kDa) may be early synthesized proteins that are polymerized into larger beta-keratins which are then packed to form larger filaments. Some proline-labeled bands differ from those produced after injection of tritiated histidine. The latter treatment does not show 10-20 kDa labeled proteins, but tends to show bands at 27, 30-33, 40-42 and 50-62 kDa. Histidine-labeled proteins mainly localize in keratohyalin-like granules and dark keratin bundles of clear-oberhautchen layers of lizard epidermis, and their composition is probably different from that of beta-keratin.
Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.
Beta-keratins constitute most of the corneous material of carapace and plastron of turtles. The production of beta-keratin in the epidermis of a turtle and tortoise (criptodirians) and of a species of pleurodiran turtle was studied after injection of tritiated proline during the growth of carapace, plastron and claws. Growth mainly occurs near hinge regions along the margins of scutes and along most of the claws (growing regions). Proline incorporation occurs mainly in the growing centers, and is more specifically associated with beta-keratin synthesis. Proline-labeled bands of protein at 12-14 kDa and 25-27 kDa, and 37 kDa, in the molecular weight range of beta-keratins, were isolated from the soft epidermis of turtles 3 h after injection of the labeled amino acid. After extraction of epidermal proteins, an antibody directed against a chicken beta-keratin was used for immunoblotting. Bands of beta-keratin at 15-17 kDa, 22-24 kDa, and 36-38 kDa appear in all species. Beta-keratin is present in the growing and compact stratum corneum of the hard (shell) and soft (limbs, neck and tail) epidermis. This was confirmed using a specific antibody against a turtle beta-keratin band of 15-16 kDa. The latter antibody recognized epidermal protein bands in the range of 15-16 kDa and 29-33 kDa, and labels beta-keratin filaments. This result indicates that different forms of beta-keratins are produced from low molecular weight precursors or that larger aggregate form during protein preparation. The present study shows that beta-keratin is abundant in the scaled epidermis of tortoise but also in the soft epidermis of pleurodiran and cryptodiran turtles, indicating that this form of hard keratin is constitutively expressed in the epidermis of chelonians.
The characteristics of scaled skin of reptiles is one of their main features that distinguish them from the other amniotes, birds and mammals. The different scale patterns observed in extant reptiles result from a long evolutive history that allowed each species to adapt to its specific environment. The present review deals with comparative aspects of epidermal keratinization in reptiles, chelonians (turtles and tortoises), lepidosaurian (lizards, snakes, sphenodontids), archosaurians (crocodilians). Initially the morphology and cytology of reptilian scales is outlined to show the diversity in the epidermis among different groups. The structural proteins (alpha-keratins and associated proteins), and enzymes utilized to form the corneous layer of the epidermis are presented. Aside cytokeratins (alpha-keratins), used for making the cytoskeleton, reptilian alpha-keratinocytes produce interkeratin (matrix) and corneous cell envelope proteins. Keratin bundles and degraded cell organelles constitute most of the corneous material of alpha-keratinocytes. Matrix, histidine-rich and sulfur-rich proteins are produced in the soft epidermis and accumulated in the cornified cell envelope. Main emphasis is given to the composition and to the evolution of the hard keratins (beta-keratins). Beta-keratins constitute the hard corneous material of scales. These small proteins are synthesized in beta-keratinocytes and are accumulated into small packets that rapidly merge into a compact corneous material and form densely cornified layers. Beta-keratins are smaller proteins (8-20 kDa) in comparison to alpha-keratins (40-70 kDa), and this size may determine their dense packing in corneocytes. Both glycine-sulfur-rich and glycine-proline-rich proteins have been so far sequenced in the corneous material of scales in few reptilian species. The latter keratins possess C- and N-amino terminal amino acid regions with sequence homology with those of mammalian hard keratins. Also, reptilian beta-keratins possess a central core with homology with avian scale/feather keratins. Multiple genes code for these proteins and their discovery and sequentiation is presently an active field of research. These initial findings however suggest that ancient reptiles already possessed some common genes that have later diversified to produce the specific keratin-associated proteins in their descendants: extant reptiles, birds and mammals. The evolution of these small proteins in lepidosaurians, chelonians and archosaurians represent the next step to understand the evolution of cornification in reptiles and derived amniotes (birds and mammals).
The first detailed histological report on reptilian skin was published well over 100 years ago (Leydig 1873). In the following 60 years a large number of studies has been accumulated and reviewed by Lange (1931). However, due to the piecemeal nature of the information available at this time, many misinterpretations occurred and no general patterns of differentiation could be demonstrated in this article.
The hard keratins of mammals and birds have been extensively studied over recent years and a considerable body of knowledge is now available on the structure and composition of the constituent proteins and of their arrangement within the keratin structure1. Mammalian keratins have a very characteristic structure in which filaments of about 70A diameter are embedded in a non-filamentous matrix composed of sulfur-rich and glycine-tryosine-rich proteins. The filaments are responsible for the α-type X-ray pattern. In contrast avian keratins contain only one family of proteins arranged as small filaments of about 30A diameter which are responsible for a characteristic X-ray pattern, often referred to as the feather type. No sequence homology has been found between avian and mammalian keratin proteins and it is generally considered that they represent separate evolutionary developments.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
The proteins of feather become soluble when they are treated with a combination of a disulphide bond-breaking reagent and a protein denaturant. The disulphide bonds of feather keratin have been cleaved by reduction with alkaline thioglycollate (Goddard and Michaelis 1934; Harrap and Woods 1964a), by sulphitolysis (Woodin 1954; Harrap and Woods 1964a) and by oxidation (Harrap and Woods 1964 a). Although detergents (Ward et al. 1946) and dissociating agents such as guanidine and urea have been tested (Jones and Mecham 1943), the customary practice is to extract into 8 M urea at pH 9.8 in the presence of either 0.1 M sodium thioglycollate (Harrap and Woods 1967) or 0.1 M β-mercaptoethanol (Kemp and Rogers 1972) and to convert the proteins to their S-carboxymethyl (SCM) derivatives by reaction with sodium iodoacetate.
The integuments of extant vertebrates display a variety of epidermal appendages whose patterns, morphology and terminal differentiation (epidermal keratins) depend upon interactions between ectodermal (epidermis) and mesodermal (dermis) tissues. In reptiles and birds, appendage morphogenesis precedes terminal differentiation. Studies have demonstrated that appendage morphogenesis influences the expression of the appendage specific keratin genes. However, little is known about the nature of the structural genes expressed by the epidermal appendages of reptiles. How pattern formation and/or appendage morphogenesis influence terminal differentiation of reptilian appendages is not known. The epidermal appendages of reptiles and birds are characterized by the presence of both alpha (α) and beta (β) type keratin proteins. Studies have focused on the genes of avian β keratins because they are the major structural proteins of feathers. The occurrence of β keratin proteins in the scales and claws of both birds and reptiles and their immunological cross-reactivity suggest that the genes for reptilian β keratins may be homologous with those of birds. In bird appendages, the β keratins are the products of a large family of homologous genes. Specific members of this gene family are expressed during the development of each appendage. Recent sequence analyses of feather β keratins, from different orders of birds, demonstrate that there is more diversity at the DNA level than was implied by earlier protein sequencing studies. Immunological techniques show that the same antibodies that react with the epidermal β keratins of the chicken (Gallus domesticus) react with the epidermal β keratins of American alligators (Alligator mississippiensis). Furthermore, a peptide sequence (20 amino acids) from an alligator claw β keratin is similar to a highly conserved region of avian claw, scale, feather, and feather-like β keratins. These observations suggest that the β keratin genes of avian epidermal appendages have homologues in the American alligator. Understanding the origin and evolution of the β keratin gene families in reptiles and birds will undoubtedly add to our understanding of the evolution of skin appendages such as scales and feathers.
The ϕ keratins extracted from individual turtles and snakes were compared. The extent and nature of the molecular differentiation associated with the production of structurally distinct epidermal tissues was determined. The electrophoretic comparisons, molecular weight, and chemical fractionation indicate that these tissues contain unique proportions of the constituent keratin monomers specific to each species. This pattern of differentiation is similar to that previously observed for avian scale, claw, and beak, and for mammalian horn and hoof. This suggests that several “scale-like” structures with distinctive chemical properties may be produced by a single individual without the synthesis of wholly unique proteins. The implications of these observations for the evolution of mammalian hair and avian feathers are discussed.
The integuments of extant vertebrates display a variety of epidermal appendages whose patterns, morphology and terminal differentiation (epidermal keratins) depend upon interactions between ectodermal (epidermis) and mesodermal (dermis) tissues. In reptiles and birds, appendage morphogenesis precedes terminal differentiation. Studies have demonstrated that appendage morphogenesis influences the expression of the appendage specific keratin genes. However, little is known about the nature of the structural genes expressed by the epidermal appendages of reptiles. How pattern formation and/or appendage morphogenesis influence terminal differentiation of reptilian appendages is not known.
The epidermal appendages of reptiles and birds are characterized by the presence of both alpha (α) and beta (β) type keratin proteins. Studies have focused on the genes of avian β keratins because they are the major structural proteins of feathers. The occurrence of β keratin proteins in the scales and claws of both birds and reptiles and their immunological cross-reactivity suggest that the genes for reptilian β keratins may be homologous with those of birds. In bird appendages, the β keratins are the products of a large family of homologous genes. Specific members of this gene family are expressed during the development of each appendage. Recent sequence analyses of feather β keratins, from different orders of birds, demonstrate that there is more diversity at the DNA level than was implied by earlier protein sequencing studies.
Immunological techniques show that the same antibodies that react with the epidermal β keratins of the chicken (Gallus domesticus) react with the epidermal β keratins of American alligators (Alligator mississippiensis). Furthermore, a peptide sequence (20 amino acids) from an alligator claw β keratin is similar to a highly conserved region of avian claw, scale, feather, and feather-like β keratins. These observations suggest that the β keratin genes of avian epidermal appendages have homologues in the American alligator. Understanding the origin and evolution of the β keratin gene families in reptiles and birds will undoubtedly add to our understanding of the evolution of skin appendages such as scales and feathers.
As surface and lining tissues, human epithelial cells play a common protective role in the body. This role is manifested by the construction of extensive cytoskeletal architecture inside each cell. The surface of the skin has perhaps the greatest need for a protective cytoskeleton. While the filaments in the cortex cells of the hair are arranged in an orderly and rigid fashion, the bundles of filaments in the squames of the epidermis appear to be more loosely packed. The different organization of cytoskeletal elements within these two types of cells may contribute to the markedly distinct packing of cells within the two tissues. In contrast to the terminally differentiated epithelial cells on the body surface, the cells of the basal layer of the epidermis as well as cells of internal epithelia must maintain their ability to divide and to undergo normal metabolic processes and cell movement. In addition to the variation in the abundance of keratin filaments, the proteins that make up these filaments are also different for different epithelia. Different pairs of keratins seem to be expressed in different epithelial cells at different stages of development and differentiation. Some of the major mysteries have been (1) to determine the ways the differential expression of different pairs of keratins might relate to cytoskeletal design and (2) to elucidate the molecular mechanisms underlying this process. Recent sequence analyses of the keratins and the genes that encode these proteins have begun to reveal the answers to these questions.
Previous reports on the fine structure of lizard epidermis are confirmed and extended by SEM and TEM observations of cell differentiation and the form of shed material from the American anole Anolis carolinensis. Attention is drawn to two issues: 1) the tips of the spinules arising from the mature oberhautchen are markedly curved; this morphology can be seen during differentiation; 2) the median keels of scales from all parts of the body show “naked” oberhautchen cells that lack characteristic spinules, but have a membrane morphology comprising a complex system of serpentine microridges.Maderson's ([1966] J. Morphol. 119:39–50) “zip-fastener” model for the role of the shedding complex formed by the clear layer and oberhautchen is reviewed and extended in the light of recent SEM data. Apparently periodic lepidosaurian sloughing permits somatic growth; understanding how the phenomenon is brought about requires integration of data from the organismic to the molecular level. The diverse forms of integumentary microornamentation (MO) reported in the literature can be understood by considering how the cellular events occurring during the renewal phase prior to shedding relate to the emergence of the form-function complex of the β-layer, which provides physical protection. Issues concerning the evolutionary origin of lepidosaurian skin-shedding are discussed. J. Morphol. 236:1–24, 1998. © 1998 Wiley-Liss, Inc.
Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation. J. Morphol. 248:64–79, 2001. © 2001 Wiley-Liss, Inc.
1.1. Claw keratin of the lizard, Varanus gouldii, is composed mainly of 13,000 proteins rich in glycine and cystine. This study deals with a tryptophan-rich group of about 20 constituent proteins comprising about one-third of the mass.2.2. As , these proteins were separated by fractional precipitation and gel filtration, then characterized by amino acid analysis, end group analysis, electrophoresis and ultracentrifugation.3.3. It is considered that the majority of lizard claw proteins are more closely allied to those of avian beak and claw than feather proteins.4.4. Claw keratin also contains minor proteins which resemble mammalian keratin high-tyrosine proteins.
Reptile keratins produce complex electrophoretic patterns, contain a number of size classes, and contain protein fractions analogous to the fractions found in other keratins. Thus, reptile keratins are similar to the heterogenous keratins of birds and mammals, and quite different from amphibian epidermal keratins. This heterogeneity may be related to the multiple functions performed by the epidermis of these organisms.
The chemical diversity of reptile keratins seems to depend on the morphological differences between the tissues in which they occur. This situation is also found among these proteins in mammals and birds suggesting that keratin diversity is related to the morphological and presumably functional differentiation of epidermal tissues. The distribution of the keratin fractions in each tissue contributes to this diversity but the significance of these fractional differences is uncertain.
A comparison of the half-cystine and glycine content of vertebrateα andØ keratins suggests that theα andØ proteins of reptiles may be related to the softα keratins of mammals and amphibians. Mammalian hard keratins probably represent a uniquely derived group of proteins which are unlike the other vertebrate keratins. The presence of a “high sulphur” matrix component in both hard mammalianα and reptilian Ø keratins may represent some form of molecular convergence which provides these distantly related proteins with similar physical or organizational properties.
In this review, the structure and biological formation of hard alpha-keratin are drawn together. The hard keratins comprising wool, hairs, quills, hooves, horns, nails and baleen contain partly alpha-helical polypeptides which show homology with epidermal polypeptides only in the helical regions. These polypeptides (about 32 chains) are organized into intermediate filaments (IFs) of 7.5 nm diameter which are embedded in variable amounts of a matrix of non-helical cystine-rich proteins and glycine-tyrosine-rich proteins. The total number of proteins may exceed 100. In addition keratins contain a variety of lipid components. Wool and hair are produced in follicles in a multistep procedure. In the lower levels of the follicle, IFs without associated matrix are found. Subsequently matrix proteins are laid down between the IFs and further synthesis takes place concurrently. Finally the proteins are insolubilized by the oxidative formation of disulphide bonds. Keratinized fibres shows considerable complexity and diversity in the structural arrangement of IFs and matrix within cortical cells. Typically the IFs show hexagonal packing or give a whorl-like appearance in cross-section.
Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms.
The precise localization of particular keratin polypeptides eventually enables to sub-classify both embryonic and adult epithelial cells and tissues with respect to their relative degree of differentiation. This capacity should be exceptionally useful in studying the various aspects of epithelial differentiation. These localization studies are largely dependent upon immunological procedures involving monoclonal antibodies. While a great deal has been learned about the biochemical and immunological properties of keratin polypeptides through the use of broadly cross-reacting monoclonal antibodies such as AE-1 and AE-3, they are of limited usefulness in the tissue localization of individual keratins within various epithelia. Any studies using conventional or monoclonal antibodies that recognize multiple keratin polypeptides must always be performed in conjunction with the immunochemical analysis of the keratins recognized in a given tissue before conclusions may be made about their localization. The current “state of the art” monoclonal antibody studies on keratin localization involve highly selective antibodies (usually recognizing a single keratin polypeptide) that demonstrate an extraordinary degree of keratin specificity. Despite the relative absence of comprehensive studies on the precise tissue localization of individual keratin polypeptides, the apparent ubiquity of the keratin pair concept and the theory of differentiation-specific pairs are so uniformly consistent that the total keratin polypeptide composition of any epithelial tissue, given only the species of origin and its histological characteristics, can be accurately predicted.
Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized histologically by absent or reduced keratohyaline granules in the epidermis and mild hyperkeratosis. The basic defect in ichthyosis vulgaris is unknown. We have tested for the presence of filaggrin and its precursor, profilaggrin, in the epidermis of affected and unaffected individuals from 2 families with ichthyosis vulgaris and correlated its presence and relative quantity with ultrastructure findings in the same individuals.
Filaggrin was present on stained sodium dodecyl sulfate gels and immunoblots of epidermal proteins from controls and unaffected family members. It was absent from the more severely affected individuals in each family and reduced in intensity in the less severely affected family members. Immunohistology in controls showed localization of filaggrin-related protein in the stratum corneum and within the granular layer. In contrast, tissue from affected individuals showed little or no reaction. Electron microscopic studies showed that keratohyaline granules were absent in 3 severely affected individuals, and reduced in number in the others. The relative amount of keratohyalin by electron microscopy correlated with the amount of filaggrin detectable on immunoblots. The stratum corneum was thicker than in normals but showed the typical “keratin pattern” staining suggesting that filaggrin is not essential for keratin filament aggregation and may have another function in vivo.
We have demonstrated that the structural proteins, profilaggrin and filaggrin, are reduced or absent in 5 patients from 2 pedigrees with ichthyosis vulgaris. This biochemical abnormality correlates with the morphologic reduction in the amount of keratohyalin, and with the clinical severity of the disorder.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Epidermal material from a variety of reptilian species, avian and mammalian scales have been examined by standard histological and x-ray diffraction techniques. It has been found that morphologic and/or tinctorial properties are not good criteria for the identification of specific fibrous protein types. The distribution of fibrous protein types in reptilian epidermal material is very variable, especially in turtles. Lepidosaurian reptiles (the tuatara, snakes and lizards) are unique in showing an alternating vertical distribution of feather and α-type proteins over the entire body surface. The protein distribution in crocodilian scales resembles exactly that found on avian scales. Mammals only possess the α-type protein whatever the nature of the epidermal structure of modification.
The outer shell of translucent keratin has been dissected from the claws of the lizard,Varanus gouldii. It is free of calcium and hydroxyproline, in contrast to the fibrous support, and contains proteins rich in glycine (28 residues %) and half-cystine (13%). These proteins have been obtained in soluble form by treatment with 2-mercaptoethanol in 8M urea at pH 11 followed by alkylation with iodoacetate to giveS-carboxymethyl kerateines. The three major components resolved by SDS polyacrylamide gel electrophoresis have been isolated by fractional precipitation with ammonium sulfate followed by chromatography on DEAE-cellulose or Sephadex. Two of the components, low in tryptophan content, appear to be homologous and are relatively homogeneous with respect to both size and charge whereas the third, a tryptophan-rich material, appears to contain about 20 different molecular species as judged by gel electrophoresis in urea at pH 8.9. The molecular weights of two of the isolated omponents (the tryptophan-rich and the major of the two tryptophanpoor components) are about 13000 as determined by equilibrium ultracentrifugation studies.
The major lizard claw proteins are therefore similar in size and glycine content to the proteins of avian beak and claw but differ in containing more cystine and less tyrosine. On the other hand, the reptilian proteins resemble the mammalian high-tyrosine proteins (Type II) in cystine content and overall amino acid composition, but differ in size with the lizard proteins being larger. It is suggested however that they are unlikely to be homologous.
We have traced the evolutionary origins of keratin-like sequences to the genomes of lower eukaryotes. The proteins encoded by these genes have evolved to form the intermediate filaments that comprise the backbone of vertebrate skin cells. Two related but distinct types of keratins encoded by two separate multigene subfamilies are expressed in the epidermal keratinocytes of vertebrate species from fish to human. Both at the level of protein and at the level of DNA, these two classes of keratins are coordinately conserved throughout vertebrate evolution, indicating the central role that both types of keratins must play in the assembly and structure of the 8-nm filament.
The development of scales was analysed in embryos of the Jamaican Iguanid lizard Anolis lineatopus after the injection of cell proliferation markers [3H]thymidine and 5-bromodeoxyuridine. Embryos were fixed at successive postinjection periods, from 2 h up to 13 d and sections of developing skin were studied by autoradiography and immunocytochemistry. The epidermis during the initial stages of morphogenesis (flat epidermis and symmetric scale stages) expands chiefly by the tangential proliferation of the basal layer. The superficial periderm took part in its own laminar expansion. The rates of proliferation in the epidermis and dermis were similar during the flat epidermal stage, but dermal proliferation decreased under the wave-like epidermis of symmetric scales. No specific localisation of proliferating cells was visible either in the epidermis and or the dermis at these 2 early stages. Scale asymmetry is brought about by cell multiplication and hypertrophy of the cells of the future outer surface of the scale that behaves as an epithelial placode capable of producing beta-keratinised cells. The growth of the outer surface determines the asymmetry of the scale. Conversely, the future inner side and hinge region showed a lower degree of cell proliferation and cells remained cuboidal or became flat. Also the proliferation of the dermis under the scaling epidermis was significantly diminished. During the asymmetric scale stage, labelled cells were more common in the hypertrophied basel epithelium from 2 h until 4 d postinjection, during which time a few cells moved into the upper keratinizing layers. At 6-13 d postinjection, labelled cells were commonly seen in the upper beta-keratinising layers.
X-ray diffraction and electron microscope studies of hard keratins (e.g. claws, scales, feathers and hair) have shown that they all have a filamentous texture but that the molecular structure of the filaments in mammalian keratins is quite different from that in avian keratins. The framework of the filaments in mammalian keratin consists of two-strand coiled coils of alpha-helices whereas the framework in avian keratins is composed of beta-sheets. Reptilian hard keratins have not been studied in detail but the X-ray diffraction pattern is very similar to that obtained from avian hard keratins leading to the supposition that the framework of the filaments is also composed of beta-sheets. The present contribution describes an analysis of the sequence of a lizard claw protein using structural probes which reveal the origins of the common structural features of the filaments in avian and reptilian keratin.
In the epidermis of lizards, alpha- and beta-keratins are sequentially produced during a shedding cycle. Using pre- and post-embedding immunocytochemistry this study shows the ultrastructural distribution of 3 alpha-keratin antibodies (AE1, AE2, AE3) in the renewing epidermis and in the shedding complex of the regenerating tail of the lizard Podarcis muralis. The AE1 antibody that recognizes acidic low MW keratins is confined to tonofilament bundles in basal and suprabasal cells but is not present in keratinizing beta- and alpha-cells. The AE2 antibody that recognises higher MW keratins weakly stains pre-keratinized cells and intensely keratinized alpha-layers. A weak labeling is present in small electrondense areas within the beta-layer. The AE3 antibody, that recognizes low and high MW basic keratins, immunolabels tonofilament bundles in all epidermal layers but intensely the alpha-keratinizing and keratinized layers (mesos, alpha-, lacunar and clear). Keratohyalin-like granules, present in the clear cells of the shedding layer, are negative to these antibodies so that the cornified clear layer contains keratins mixed with non-keratin material. The AE3 antibody shows that the mature beta-layer and the spinulated folds of the oberhautchen are labeled only in small dense areas among the prevalent electron-pale beta-keratin material. Therefore, some alpha-keratin is still present in the beta-layer, and supports the idea that alpha-keratins (basic) function as scaffold for beta-keratin deposition.
Using immunocytochemistry at light- and electron-microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian alpha-keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and beta-layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized beta- and alpha-layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the beta-layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron-dense areas within the beta-layer, suggesting retention of alpha-keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40-41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77-78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed.
The distribution of three anti-cytokeratin (α-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external β-layer, an underlying α-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized β- and α-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the α-layer and gives a lower reaction in the β-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the α-layer, and absent from the β-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep α-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense β-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the α- and the β-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.
Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. They exist as highly dynamic networks of cytoplasmic 10-12 nm filaments that are obligate heteropolymers involving type I and type II keratins. The primary function of keratins is to protect epithelial cells from mechanical and nonmechanical stresses that result in cell death. Other emerging functions include roles in cell signaling, the stress response and apoptosis, as well as unique roles that are keratin specific and tissue specific. The role of keratins in a number of human skin, hair, ocular, oral and liver diseases is now established and meshes well with the evidence gathered from transgenic mouse models. The phenotypes associated with defects in keratin proteins are subject to significant modulation by functional redundancy within the family and modifier genes as well. Keratin filaments undergo complex regulation involving post-translational modifications and interactions with self and with various classes of associated proteins.
Beta (beta) keratins are present only in the avian and reptilian epidermises. Although much is known about the biochemistry and molecular biology of the beta keratins in birds, little is known for reptiles. In this study we have examined the distribution of beta keratins in the adult epidermis of turtle, lizard, snake, tuatara, and alligator using light and electron immunocytochemistry with a well-characterized antiserum (anti-beta(1) antiserum) made against a known avian scale type beta keratin. In lizard, snake, and tuatara epidermis this antiserum reacts strongly with the beta-layer, more weakly with the oberhautchen before it merges with the beta-layer, and least intensely with the mesos layer. In addition, the anti-beta(1) antiserum reacts specifically with the setae of climbing pads in gekos, the plastron and carapace of turtles, and the stratum corneum of alligator epidermis. Electron microscopic studies confirm that the reaction of the anti-beta(1) antiserum is exclusively with characteristic bundles of the 3-nm beta keratin filaments in the cells of the forming beta-layer, and with the densely packed electron-lucent areas of beta keratin in the mature bet- layer. These immunocytochemical results suggest that the 3-nm beta keratin filaments of the reptilian integument are phylogenetically related to those found in avian epidermal appendages.
The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (beta) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (beta) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of "four-winged" dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), "feathered feet." We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the beta keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High-line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers.
Little is known about specific proteins involved in keratinization of the epidermis of snakes, which is composed of alternating beta- and alpha-keratin layers. Using immunological techniques (immunocytochemistry and immunoblotting), the present study reports the presence in snake epidermis of proteins with epitopes that cross-react with certain mammalian cornification proteins (loricrin, filaggrin, sciellin, transglutaminase) and chick beta-keratin. alpha-keratins were found in all epidermal layers except in the hard beta- and alpha-layers. beta-keratins were exclusively present in the oberhautchen and beta-layer. After extraction and electrophoresis, alpha-keratins of 40-67 kDa in molecular weights were found. Loricrin-like proteins recorded molecular weights of 33, 50, and 58 kDa; sciellin, 55 and 62 kDa; filaggrin-like, 52 and 65 kDa; and transglutaminase, 45, 50, and 56 kDa. These results suggest that alpha-layers of snake epidermis utilize proteins with common epitopes to those present during cornification of mammalian epidermis. The beta-keratin antibody on extracts from whole snake epidermis showed a strong cross-reactive band at 13-16 kDa. No cross-reactivity was seen using an antibody against feather beta-keratin, indicating absence of a common epitope between snake and feather keratins.
During scale regeneration in lizard tail, an active differentiation of beta-keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard beta-keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3'-rapid amplification of cDNA ends analysis, 3'-RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5'-RACE. The 835-nucleotide cDNA sequence encodes a glycine-proline-rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine-glycine-X and glycine-X amino acid sequences are localized near the N-amino and C-terminal regions. The protein has the central amino acid region similar to that of claw-feather, whereas the head and tail regions are similar to glycine-tyrosine-rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating beta-layers of the regenerating scales. The synthesis of beta-keratin from its mRNA occurs among ribosomes or is associated with the surface of beta-keratin filaments.
The structural proteins of hair: isolation, characterization and regulation of biosynthesis In: GoldsmithL, editor. Physiology, biochemistry and molecular biology of the skin
- Jm Gillespie
- Jm Gillespie
Molecular and cellular biology of keratins Physiology, biochemistry and molecular biology of the skin
- Steinert Pm
- Freedberg
- Im
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