FIG 1 - uploaded by Jeroen Bakkers
Content may be subject to copyright.
Radiolabeling of gastrulation-specific metabolites. Extracts of zebrafish or carp embryos from the gastrulation stage were incubated in the presence of radiolabeled or unlabeled UDP-GlcNAc (see Materials and Methods). (A) Incorporation of UDP-[U 14 C]GlcNAc into HPLC fractions with retention times similar to chitin tetraose (striped box) and chitin pentaose (filled box). In the incubations where unlabeled UDP-GlcNAc was used, the equivalent fractions were incubated with the NodZ protein in the presence of GDP-[U14 C]fucose. By using this assay it is possible to specifically detect chitin oligosaccharides at concentrations as low as 1 picomol (data not shown). The pooled fractions were used for chitinase and chitobiase treatments and separated on TLC (B-D). (B) Radiolabeling of metabolites obtained from carp embryos and separated by HPLC, using the NodZ transfucosylation assay. Lanes: 1, fucosylated chitin oligosaccharide standards (as described in ref. 4); 2 and 3, HPLC fractions with retention times similar to chitin tetraose after transfu
Source publication
Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation...
Contexts in source publication
Context 1
... that in vitro zebrafish embryos of the gastrulation stage incorporate radioactive uridine 5-diphospho-N-acetyl-D- [U 14 C]glucosamine (UDP-[U 14 C]GlcNAc) into metabolites that in HPLC analysis have retention times similar to that of chitin pentaose (-1,4-linked GlcNAc) (9). Using gastrula- tion stage embryos of carp we have obtained similar data (Fig. 1A). We also treated HPLC fractions with purified NodZ protein of Bradyrhizobium. This enzyme is an -1,6- fucosyltransferase with substrate specificity for chitin oligo- saccharides that can be used for in vitro transfucosylation (4). Metabolites synthesized by cell extracts of zebrafish and carp embryos from the gastrulation stage were ...
Context 2
... (4). Metabolites synthesized by cell extracts of zebrafish and carp embryos from the gastrulation stage were purified by HPLC (see Materials and Methods). HPLC fractions having reten- tion times similar to standards of chitin tetraose and chitin pentaose were used as substrates in the transfucosylation assay with radiolabeled GDP-[U-14 C]fucose (Fig. 1B). The results show that these fractions contain metabolites that can be fucosylated efficiently by the NodZ protein in the pres- ence of GDP-[U-14 C]fucose. The fucosylated derivatives of the metabolites in HPLC fractions with retention times similar to chitin tetraose and chitin pentaose (Fig. 1 A) migrate on TLC as fucosylated chitin ...
Context 3
... assay with radiolabeled GDP-[U-14 C]fucose (Fig. 1B). The results show that these fractions contain metabolites that can be fucosylated efficiently by the NodZ protein in the pres- ence of GDP-[U-14 C]fucose. The fucosylated derivatives of the metabolites in HPLC fractions with retention times similar to chitin tetraose and chitin pentaose (Fig. 1 A) migrate on TLC as fucosylated chitin tetraose (Fig. 1B, lane 3) or fucosylated chitin pentaose (Fig. 1 B, lane 5, and C, lanes 1 and 3), respectively. When these fucosylated mate- rials were treated with a combination of chitinase and chitobiase, a degradation pattern typical for chitin oligosac- charides is found (Fig. 1B, lanes 2 and ...
Context 4
... The results show that these fractions contain metabolites that can be fucosylated efficiently by the NodZ protein in the pres- ence of GDP-[U-14 C]fucose. The fucosylated derivatives of the metabolites in HPLC fractions with retention times similar to chitin tetraose and chitin pentaose (Fig. 1 A) migrate on TLC as fucosylated chitin tetraose (Fig. 1B, lane 3) or fucosylated chitin pentaose (Fig. 1 B, lane 5, and C, lanes 1 and 3), respectively. When these fucosylated mate- rials were treated with a combination of chitinase and chitobiase, a degradation pattern typical for chitin oligosac- charides is found (Fig. 1B, lanes 2 and 4; and D, lanes 2 and 3). From these results we conclude that ...
Context 5
... metabolites that can be fucosylated efficiently by the NodZ protein in the pres- ence of GDP-[U-14 C]fucose. The fucosylated derivatives of the metabolites in HPLC fractions with retention times similar to chitin tetraose and chitin pentaose (Fig. 1 A) migrate on TLC as fucosylated chitin tetraose (Fig. 1B, lane 3) or fucosylated chitin pentaose (Fig. 1 B, lane 5, and C, lanes 1 and 3), respectively. When these fucosylated mate- rials were treated with a combination of chitinase and chitobiase, a degradation pattern typical for chitin oligosac- charides is found (Fig. 1B, lanes 2 and 4; and D, lanes 2 and 3). From these results we conclude that extracts of zebrafish and carp embryos of the gastrulation stage produce ...
Context 6
... and chitin pentaose (Fig. 1 A) migrate on TLC as fucosylated chitin tetraose (Fig. 1B, lane 3) or fucosylated chitin pentaose (Fig. 1 B, lane 5, and C, lanes 1 and 3), respectively. When these fucosylated mate- rials were treated with a combination of chitinase and chitobiase, a degradation pattern typical for chitin oligosac- charides is found (Fig. 1B, lanes 2 and 4; and D, lanes 2 and 3). From these results we conclude that extracts of zebrafish and carp embryos of the gastrulation stage produce chitin oligosaccharides in vitro. A clear difference between the two is that carp embryos predominantly produce chitin tetrasac- charides (Fig. 1B, compare lanes 3 and 5), whereas zebrafish embryos produce ...
Context 7
... typical for chitin oligosac- charides is found (Fig. 1B, lanes 2 and 4; and D, lanes 2 and 3). From these results we conclude that extracts of zebrafish and carp embryos of the gastrulation stage produce chitin oligosaccharides in vitro. A clear difference between the two is that carp embryos predominantly produce chitin tetrasac- charides (Fig. 1B, compare lanes 3 and 5), whereas zebrafish embryos produce both chitin tetrasaccharides and pentasac- charides ( Figs. 1 A and 2A). (Fig. 2 A). Samples that were first treated with a purified chitinase do not induce an alkalinization response. By comparing the pH shift induced by the zebrafish chitin oligosaccharides and a dilution series of chitin ...
Context 8
... these results we conclude that extracts of zebrafish and carp embryos of the gastrulation stage produce chitin oligosaccharides in vitro. A clear difference between the two is that carp embryos predominantly produce chitin tetrasac- charides (Fig. 1B, compare lanes 3 and 5), whereas zebrafish embryos produce both chitin tetrasaccharides and pentasac- charides ( Figs. 1 A and 2A). (Fig. 2 A). ...
Similar publications
Properties of chitin synthase obtained from mycelial homogenates of Mortierella pusilla and Mortierella candelabrum were compared. The enzyme was localized mainly in the mixed membrane fractions of both species. The reaction product, chitin, was characterized by its insolubility in weak acid and alkali and by the release of glucosamine and diacetyl...
Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1...
Simple cladogenetic theory suggests that gene genealogies can be used to detect mixis in a population and delineate reproductively isolated groups within sexual taxa. We have taken this approach in a study of Coccidioides immitis, an ascomycete fungus responsible for a recent epidemic of coccidioidomycosis (Valley fever) in California. To test whet...
Chitin is among the most important components of the crustacean cuticular exoskeleton and intestinal peritrophic matrix. With the progress of genomics and sequencing technology, a large number of gene sequences related to chitin metabolism have been deposited in the GenBank database in recent years. Here, we summarized the genes and pathways associ...
RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical pierc...
Citations
... In mammals, GlcNAc and GlcN are components of glycoproteins, proteoglycans, glycosaminoglycans (GAGs) and other connective tissue building blocks [15][16][17][18][19][20]. Despite being a building block of biomacromolecules, GlcNAc seldom exists in free form, except in human milk (at 600-1500 mg/mL) [44,45]. Because GlcNAc not only play a role in plant organogenesis and invertebrate embryogenesis [46] but also has therapeutic potential in the treatment of a wide variety of diseases, economic feed-stocks and high efficiency processes for producing GlcNAc are urgently needed in industry. ...
... contrarily, some research data have indicated that chitin oligosaccharides are important for embryogenesis in vertebrates, whereas chitin synthase genes are present in numerous fishes and amphibians [37,38]. In this study, chitinase was conserved in five species; however, chitin synthase was only found in the zebrafish and frog genomes (Additional file 8: Table S7). ...
Background:
Glucose plays a key role as an energy source in most mammals, but its importance in fish appears to be limited that so far seemed to belong to diabetic humans only. Several laboratories worldwide have made important efforts in order to better understand this strange phenotype observed in fish. However, the mechanism of carbohydrate/glucose metabolism is astonishingly complex. Why basal glycaemia is different between fish and mammals and how carbohydrate metabolism is different amongst organisms is largely uncharted territory. The utilization of comparative systems biology with model vertebrates to explore fish metabolism has become an essential approach to unravelling hidden in vivo mechanisms.
Results:
In this study, we first built a database containing 791, 593, 523, 666 and 698 carbohydrate/glucose metabolic genes from the genomes of Danio rerio, Xenopus tropicalis, Gallus gallus, Mus musculus and Homo sapiens, respectively, and most of these genes in our database are predicted to encode specific enzymes that play roles in defined reactions; over 57% of these genes are related to human type 2 diabetes. Then, we systematically compared these genes and found that more than 70% of the carbohydrate/glucose metabolic genes are conserved in the five species. Interestingly, there are 4 zebrafish-specific genes (si:ch211-167b20.8, CABZ01043017.1, socs9 and eif4e1c) and 1 human-specific gene (CALML6) that may alter glucose utilization in their corresponding species. Interestingly, these 5 genes are all carbohydrate regulation factors, but the enzymes themselves are involved in insulin regulation pathways. Lastly, in order to facilitate the use of our data sets, we constructed a glucose metabolism database platform ( http://101.200.43.1:10000/ ).
Conclusions:
This study provides the first systematic genomic insights into carbohydrate/glucose metabolism. After exhaustive analysis, we found that most metabolic genes are conserved in vertebrates. This work may resolve some of the complexities of carbohydrate/glucose metabolic heterogeneity amongst different vertebrates and may provide a reference for the treatment of diabetes and for applications in the aquaculture industry.
... Cell surface glycans contain complex information that when decoded by specific carbohydrate-binding proteins, can determine the nature and outcome of interactions between cells, or cells and the extracellular matrix (ECM). Among these proteins, the galectins [1][2][3] constitute a structurally conserved family of animal lectins defined by their affinity for β-galactosides, and a characteristic sequence motif in the carbohydrate recognition domain (CRD) [4,5]. Galectins are widely distributed in eukaryotic taxa, and their early emergence in evolution has been revealed by the presence of a protein with the galectin fold in the protistan parasite Toxoplasma gondii, and galectin-like proteins in the fungus Coprinopsis cinerea and in the sponge Geodia cydonium [6][7][8]. ...
Galectins constitute a conserved and widely distributed lectin family characterized by their binding affinity for ß-galactosides and a unique binding site sequence motif in the carbohydrate recognition domain (CRD). In spite of their structural conservation, galectins display a remarkable functional diversity, by participating in developmental processes, cell adhesion and motility, regulation of immune homeostasis, and recognition of glycans on the surface of viruses, bacteria and protozoan parasites. In contrast with mammals, and other vertebrate and invertebrate taxa, the identification and characterization of bona fide galectins in aquatic mollusks has been relatively recent. Most of the studies have focused on the identification and domain organization of galectin-like transcripts or proteins in diverse tissues and cell types, including hemocytes, and their expression upon environmental or infectious challenge. Lectins from the eastern oyster Crassostrea virginica, however, have been characterized in their molecular, structural and functional aspects and some notable features have become apparent in the galectin repertoire of aquatic mollusks. These including less diversified galectin repertoires and different domain organizations relative to those observed in vertebrates, carbohydrate specificity for blood group oligosaccharides, and up regulation of galectin expression by infectious challenge, a feature that supports their proposed role(s) in innate immune responses. Although galectins from some aquatic mollusks have been shown to recognize microbial pathogens and parasites and promote their phagocytosis, they can also selectively bind to phytoplankton components, suggesting that they also participate in uptake and intracellular digestion of microalgae. In addition, the experimental evidence suggests that the protozoan parasite Perkinsus marinus has co-evolved with the oyster host to be selectively recognized by the oyster hemocyte galectins over algal food or bacterial pathogens, thereby subverting the oyster's innate immune/feeding recognition mechanisms to gain entry into the host cells.
Copyright © 2015. Published by Elsevier Ltd.
... Specifically, compared with the control group, DAC suture tended to enhance much smoother re-epithelialization. Recent evidence points to DG42 protein and hyaluronan. 41,42 The former can produce chitooligomers acting as templates for hyaluronan synthesis. As a result, the high concentration of hyaluronan devotes to the correct tissue reconstitution of the wounds in the fetus heal. ...
Diacetyl chitin (DAC) is an acidylated chitin obtained using acetic anhydride mixed perchloric acid system. By wet spinning and weaving technique, DAC has been successfully developed into a novel absorbable surgical suture. Thanks to the unique properties of chitins, the potential application of this novel monocomponent multifilament DAC suture may break the monopoly of synthetic polymer sutures in wound closure area. In this study, DAC was synthesized and characterized by multiple approaches including elemental analysis, Fourier transform infrared spectrometry (FTIR), and X-ray diffraction (XRD). In addition, we performed the feasibility assessment of DAC suture (USP 2-0) as absorbable suture for wound healing. Several lines of evidences suggested that DAC suture had comparable mechanical properties as synthetic polymer sutures. Moreover, DAC suture retained approximately 63% of the original strength at 14 days and completely absorbed in 42 days with no remarkable tissue reaction in vivo. Most important of all, DAC suture significantly promoted skin regeneration with faster tissue reconstruction and higher wound breaking strength on a linear incisional wound model. All these results demonstrated the potential use of DAC suture in short- or middle-term wound healing, such as epithelial and connective tissue. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.
© 2015 Wiley Periodicals, Inc.
... One GH protein, YKL-40, is a growth factor that induces cell proliferation by activating protein kinase signalling and YKL-40 over-expression enhances the migration of human macrophage and endothelial cells [22,23]. In contrast to mammals, cyprinid fish not only appear to synthesise chitin oligosaccharides de novo, but chitooligosacharides also appear to play a role in their early development [14,24]. Chitin recognition leads to induction of innate immunity and there are reports that medium sized chitin macro-particles (but not small-sized particles) may be linked to a possible role for these chitin particles in asthma and allergy pathogenesis [25]. ...
... Zebrafish might be a useful system to test LCO biological activity and tissue specificity in a vertebrate model. This is because (a) zebrafish produce endogenous chito-oligosaccharides that appear to play a role in early embryogenesis [14,24] (although this is not demonstrated in mammals) and (b) zebra fish LysM domain and chitinase proteins show specific expression patterns in the nervous system during embryogenesis [19] and the accessibility of embryogenesis in this system has distinct advantages. ...
Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro-or
... Chitosan also entraps growth factors for acceleration of the healing; limitation of scar formation and retraction [200,201]. Recent evidence points to the DG42 protein, a chito oligomer synthase that during embryogenesis produces chito oligomers acting as primers in the synthesis of hyaluronan [202]. Most preparations of hyaluronan have chito oligomers at their reducing end, that act as templates for hyaluronan synthesis and the high concentration of hyaluronan helps in healing the wounds without scar formation [203]. ...
... In spite of their recognized broad impact on immune responses in mammals, and the possible involvement in early embryonic development of trunk and tail in zebrafish (Bakkers et al., 1997;Semino and Allende, 2000), information about the functions and mechanisms of chitinases in early embryonic development is scarce. We clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to Chi3 being a newly-identified factor involved in the antifungal activity. ...
... Mammalian chitinases/CLPs have been implicated in several different innate and acquired immune responses, as well as tissue remodelling and fibrosis, initiated by both chitinous and non-chitinous stimulants (Johansen, 2006;Lee et al., 2009;Malaguarnera et al., 2006;Reese et al., 2007;van Eijk et al., 2005;Zhu et al., 2004). In developing zebrafish larvae, chitin oligomers and the activity of one or more, as yet unidentified, chitinase(s), have been implicated in the normal development of trunk and tail (Bakkers et al., 1997;Semino and Allende, 2000). ...
... At the maternal to zygotic transition, at the onset of epiboly, expression decreases to a level similar to CHIA.5 and CHIA.6. If indeed a chitinase, through some interplay with endogenous substrates, is implicated in the early embryonic developmental processes, as suggested by the studies of Bakkers and co-workers (Bakkers et al., 1997) and those of Semino and Allende (2000), CHIA.4 seems the strongest candidate for such a function. ...
Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1-6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development. CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels. Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis, on which functional analysis in older larvae may be founded.
... In the last two decades, these compounds have been the subject of intense interest once their versatile biological functions and activities were identified [6,7]. They have been shown to play roles in plant organogenesis and invertebrate embryogenesis [8]. The addition of chitin to soil can reduce the populations of fungal plant pathogens [9] and plant parasitic nematodes by increasing the populations of chitinolytic bacteria (i.e., bacteria with chitinases) [10][11][12]. ...
The versatile oligosaccharide biopolymers, chitin and chitosan, are typically produced using enzymatic processes. However, these processes are usually costly because chitinases and chitosanases are available in limited quantities. Fortunately, a number of commercial enzymes can hydrolyze chitin and chitosan to produce long chain chitin or chitosan oligosaccharides. Here, a platform to screen for enzymes with chitinase and chitosanase activities using a single gel with glycol chitin or glycol chitosan as a substrate was applied. SDS-resistant chitinase and chitosanase activities were observed for pancreatin. Its chitotriosidase had an optimal hydrolysis pH of 4 in the substrate specificity assay. This activity was thermally unstable, but independent of 2-mercaptoethanol. This is the first time a chitotriosidase has been identified in the hog. This finding suggests that oligochitosaccharides can be mass-produced inexpensively using pancreatin.
... The synthesis of these Nod-like structures was shown to be restricted to very early stages of development and was tied to the presence of a zebrafish homolog of the Xenopus developmental gene DG42 [51]. These oligosaccharides appear to have roles in early development as disruption of their synthesis leads to gastrulation defects [52,53]. At least some of these effects appear to be due to the ability of the chitin oligosaccharides to activate ERK pathways [54]. ...
Zebrafish (Danio rerio) remains a versatile model organism for the investigation of early development and organogenesis, and has emerged as a valuable platform for drug discovery and toxicity evaluation [1–6]. Harnessing the genetic power and experimental accessibility of this system, three decades of research have identified key genes and pathways that control the development of multiple organ systems and tissues, including the heart, kidney, and craniofacial cartilage, as well as the hematopoietic, vascular, and central and peripheral nervous systems [7–31]. In addition to their application in large mutagenic screens, zebrafish has been used to model a variety of diseases such as diabetes, polycystic kidney disease, muscular dystrophy and cancer [32–36]. As this work continues to intersect with cellular pathways and processes such as lipid metabolism, glycosylation and vesicle trafficking, investigators are often faced with the challenge of determining the degree to which these pathways are functionally conserved in zebrafish. While they share a high degree of genetic homology with mouse and human, the manner in which cellular pathways are regulated in zebrafish during early development, and the differences in the organ physiology, warrant consideration before functional studies can be effectively interpreted and compared with other vertebrate systems. This point is particularly relevant for glycosylation since an understanding of the glycan diversity and the mechanisms that control glycan biosynthesis during zebrafish embryogenesis (as in many organisms) is still developing.
Nonetheless, a growing number of studies in zebrafish have begun to cast light on the functional roles of specific classes of glycans during organ and tissue development. While many of the initial efforts involved characterizing identified mutants in a number of glycosylation pathways, the use of reverse genetic approaches to directly model glycosylation-related disorders is now increasingly popular. In this review, the glycomics of zebrafish and the developmental expression of their glycans will be briefly summarized along with recent chemical biology approaches to visualize certain classes of glycans within developing embryos. Work regarding the role of protein-bound glycans and glycosaminoglycans (GAG) in zebrafish development and organogenesis will also be highlighted. Lastly, future opportunities and challenges in the expanding field of zebrafish glycobiology are discussed.
