Available via license: CC BY 4.0
Content may be subject to copyright.
Isolation and Characterization of a Peptide Isomerase from Funnel Web Spider Venom
Abstract
A novel peptide isomerase was purified from the venom of funnel web spider, Agelenopsis aperta. The complete primary structure of the isomerase has been established by sequence analyses of polypeptide chains, assignments of disulfide bridges, carbohydrate analyses, and mass spectrometry of sugar chains. The isomerase was found to be a 29-kDa polypeptide that consists of an 18-residue light chain and a 243-residue heavy chain connected by a single disulfide bridge. The heavy chain contains three intramolecular disulfide bridges and one N-linked oligosaccharide chain with a simple trimannosyl core structure. A sequence homology search showed a significant similarity of the enzyme with serine proteases, particularly around a putative catalytic triad of the isomerase. The isomerase specifically interconverts the configuration of Ser46 of a 48-amino-acid peptide, omega-agatoxin-TK, and the conversion rate from L-Ser to D-Ser was approximately two times faster than the reverse reaction.
... Second, relatively little is known about the l-/d-isomerases responsible for DAACP biosynthesis (1). Although several reports have studied chromatographic fractions containing isomerase activity (5,(46)(47)(48) and the sequences of two isomerases have been proposed (49,50), the relative lack of information on these enzymes has slowed progress in characterizing DAACP signaling across animals. Third, identifying the receptors for bioactive peptides remains a significant challenge (51,52). ...
... After incubation, homogeneous time-resolved fluorescence was read using a Biotek Synergy Neo2 plate reader, as described above. Data were fit and log[EC 50 ] values calculated using the same procedure described above for IP1 accumulation assays. Experiments with average intra-assay coefficient of variation between technical replicates of <25% were used to calculate mean log[EC 50 ] and SD for independent experiments. ...
The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.
... The occurrence of D-configured amino acid is a rare and unique posttranslational modification in the genetically coded and ribosomal synthesized peptide natural products [49][50][51][52][53]. Venomous species such as cone snail, spider, and platypus indeed harbor this unusual modification in secretory peptide toxins [5,[9][10][11][12][13][14]16,54,55]. The reversal of configuration of amino acid in the gene product occurs under the action of an enzyme epimerase which facilitates abstraction/delivery of proton at Cα atom through two base mechanism [56,57]. Thus, the occurrence of this modification at the expense of metabolic energy bears a special significance in the structure, function, and proteolytic stability of secretory peptide natural products. ...
Distinct differences have been observed between L-tryptophan and D-tryptophan containing contryphan-Ar1131 in oxidative folding, trypsin binding, and photostabilization activity on avobenzone. [W⁵] contryphan-Ar1131 and [w⁵] contryphan-Ar1131 were chemically synthesized and characterized using RP-HPLC and mass spectrometry. Structural differences due to the change of configuration of tryptophan were evident from the optimized structures of contryphan-Ar1131 using density functional theory (DFT). The comparison of early events of oxidative folding has revealed the role of D-tryptophan in accelerating the formation of a disulfide bond. The optimized structures of the reduced form of peptides revealed the occurrence of aromatic-aromatic and aromatic-proline interactions in [w⁵] contryphan-Ar1131 which may be critical in aiding the oxidative folding reaction. The presence of the Lys6-Pro7 peptide bond indicates that contryphan-Ar1131 is resistant but may bind to trypsin allowing to assign the binding affinity of peptides to the protein surface. Competitive binding studies and molecular docking along with molecular dynamic (MD) simulations have revealed that [w⁵] contryphan-Ar1131 has more affinity for the active site of trypsin. Given tryptophan is a photostabilizer of FDA-approved chemical UV-A filter avobenzone, the report has compared the photostabilization activity of [W⁵]/ [w⁵] contryphan-Ar1131 on avobenzone under natural sunlight. [w⁵] contryphan-Ar1131 has better photostabilization activity than that of [W⁵] contryphan-Ar1131 and also individual D-tryptophan and L-tryptophan amino acids. These biochemical studies have highlighted the significance of D-tryptophan in contryphan-Ar1131 and its photostabilization activity on avobenzone may find applications in cosmetics.
... Notably, glycosylation was also reported in PTMs regulatory enzymes as isomerases [89,90]. Also, Souza et al. [41] identified several phosphorylation and glycosylation modulatory enzymes (e.g., kinase, phosphatase, glycosidase, and glycosyltransferase) that undergo glycosylation in P. paulista wasp venom. ...
Abstract Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study “PTM-functional-venomics”, herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.
... Proteins with a potential function in the venom gland include protein disulphide isomerases [107], a peptide isomerase [153], carboxypeptidases [107], and the recently identified 28 kDa serine proteases responsible for propeptide cleavage of immature toxin precursors (see next chapter). Other proteins, such a leucine-rich repeat domain-containing protein or a tachylectin 5A-like protein, showing similarity to immune active proteins of spider-related animals, were hypothesized to possibly be involved in protection of the venom gland against microbial infections [107]. ...
This review gives an overview on the development of research on spider venoms with a focus on structure and function of venom components and techniques of analysis. Major venom component groups are small molecular mass compounds, antimicrobial (also called cytolytic, or cationic) peptides (only in some spider families), cysteine-rich (neurotoxic) peptides, and enzymes and proteins. Cysteine-rich peptides are reviewed with respect to various structural motifs, their targets (ion channels, membrane receptors), nomenclature, and molecular binding. We further describe the latest findings concerning the maturation of antimicrobial, and cysteine-rich peptides that are in most known cases expressed as propeptide-containing precursors. Today, venom research, increasingly employs transcriptomic and mass spectrometric techniques. Pros and cons of venom gland transcriptome analysis with Sanger, 454, and Illumina sequencing are discussed and an overview on so far published transcriptome studies is given. In this respect, we also discuss the only recently described cross contamination arising from multiplexing in Illumina sequencing and its possible impacts on venom studies. High throughput mass spectrometric analysis of venom proteomes (bottom-up, top-down) are reviewed.
Difficult drug targets are becoming the normal course of business in drug discovery, sometimes due to large interacting surfaces or only small differences in selectivity regions. For these, a different approach is merited: compounds lying somewhere between the small molecule and the large antibody in terms of many properties including stability, biodistribution and pharmacokinetics. Venoms have evolved over millions of years to be complex mixtures of stable molecules derived from other somatic molecules, the stability comes from the pressure to be ready for delivery at a moment's notice. Snakes, spiders, scorpions, jellyfish, wasps, fish and even mammals have evolved independent venom systems with complex mixtures in their chemical arsenal. These venom-derived molecules have been proven to be useful tools, such as for the development of antihypotensive angiotensin converting enzyme (ACE) inhibitors and have also made successful drugs such as Byetta® (Exenatide), Integrilin® (Eptifibatide) and Echistatin. Only a small percentage of the available chemical space from venoms has been investigated so far and this is growing. In a new era of biological therapeutics, venom peptides present opportunities for larger target engagement surface with greater stability than antibodies or human peptides. There are challenges for oral absorption and target engagement, but there are venom structures that overcome these and thus provide substrate for engineering novel molecules that combine all desired properties. Venom researchers are characterising new venoms, species, and functions all the time, these provide great substrate for solving the challenges presented by today's difficult targets.
The discovery of enzyme-derived d-amino acid-containing peptides (DAACPs) that have physiological importance in the metazoan challenges previous assumptions about the homochirality of animal proteins while simultaneously revealing new analytical challenges in the structural and functional characterization of peptides. Most known DAACPs have been identified though laborious activity-guided purification studies or by homology to previously identified DAACPs. Peptide characterization experiments are increasingly dominated by high throughput mass spectrometry-based peptidomics, with stereochemistry rarely considered due to the technical challenges of identifying l/d isomerization. This review discusses the prevalence of enzyme-derived DAACPs among animals and the physiological consequences of peptide isomerization. Also highlighted are the analytical methods that have been applied for structural characterization/discovery of DAACPs, including results of several recent studies using non-targeted discovery methods for revealing novel DAACPs, strongly suggesting that more DAACPs remain to be uncovered.
In diesem Kapitel werden die Invertebraten-Protein- und -Peptidhormone vorgestellt. Bekannt sind Hormone vor allem von Schnecken, Insekten und Krebstieren. Zusätzlich zu den Regelungen bei Vertebraten geht es um die hormonelle Steuerung der Häutung und der Verpuppung.
Three new linear peptides containing d-leucine, named whitmantides A-C (1-3), were isolated from the dried whole bodies of Whitmania pigra Whitman. Their structures with absolute configurations were elucidated by Edman degradation, mass spectrometry, Marfey's analysis, and solid-phase synthesis. It is the first time that peptides containing d-amino acid in leeches were discovered. Compounds 1-3 displayed neuroprotective activities against oxygen-glucose deprivation/reperfusion injury on Neuro-2a cells. In addition, ex vivo serum stability tests showed that 1-3 were resistant to protease degradation.
We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with K(m) values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.
The complete primary structure of reduced and S-carboxy-methylated bovine erythrocyte superoxide dismutase has been derived through analysis of peptides from peptic, plasmin,
and hydroxylamine digests of the intact polypeptide chain, and from chymotryptic, subtilisin, and dilute acid digests of derived
peptides. From these data, the following unique amino acid sequence of 151 residues was deduced, corresponding to a molecular
weight of 15,600, in each of the 2 apparently identical subunits of the protein molecule. (Cmc denotes S-carboxymethylcysteine.)
[see PDF for equation]
We have isolated and analyzed the gene for batroxobin, a thrombin-like snake venom enzyme. Three overlapping DNA segments containing the entire batroxobin gene were identified. Sequence analysis revealed that the batroxobin gene spans 8 kilobase pairs and contains five exons. Mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of batroxobin, His-41, Asp-86, and Ser-178, are encoded by separate exons, exons 2, 3, and 5, respectively. The exon/intron organization of the batroxobin gene is different from that of the prothrombin gene but very similar to those of the trypsin and kallikrein genes. These results indicate that batroxobin is not a member of the prothrombin family but one of the trypsin/kallikrein family. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.
A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.
A TCEP (tris(2-carboxyethyl)phosphine)-based reduction/cysteine amidoalkylation strategy was utilized to solve the disulfide structures of omega-agatoxins IVB (1) and TVC (2). These p-type calcium channel antagonists, isolated from the American funnel-web spider Agelenopsis aperta, were found to have the same amino acid sequence and disulfide bond motif. The difference between omega-Aga IVB (1) and IVC (2) resides in the C-termini (Ser(46)) of both peptides. omega-Aga IVB (1) contains a D-serine residue while omega-Aga TVC (2) has an L-serine in this position. The existence of D-amino acids in eucaryotic systems is extremely rare. To our knowledge, however, this is the first time that a peptide sequence with an established cystine pattern possesses an amino acid in both D and L configurations.
The retention time for cis-trans isomerization around peptide bonds containing the imide nitrogen of prolyl residues is on the time scale of separation of such proline-containing peptides by HPLC. As a result, when a secondary equilibrium distribution of the cis and trans isomers in the chromatographic column occurs in addition to the primary distribution of the species between mobile and stationary phases, additional band spreading and distortion of peak shape occurs. On the basis of previous theoretical work, the first and second moments of the elution profiles obtained with proline peptides were employed to evaluate the rate constants for cis-trans isomerization of L-alanyl-L-proline and L-valyl-L-proline on the stationary phase surface by using the appropriate rate constants for isomerization in the eluent proper.
This chapter presents a procedure for nonenzymic cleavage of proteins with hydroxylamine, which provides a relatively specific means of producing large peptide fragments suitable for further chemical analysis. Cleavage occurs at Asn-Gly bonds and results from the tendency of the asparaginyl side chain to cyclize, forming a substituted succinimide that is susceptible to nucleophilic attack by hydroxylamine. The increased susceptibility of Asn-Gly bonds in comparison with other asparaginyl bonds may result from the greater ease with which the asparaginyl side chain can cyclize in the absence of steric hindrance imposed by a side chain on the succeeding amino acid. The extent of cleavage achieved varies with the protein; cleavage is enhanced by complete denaturation of the protein and by the use of 6 M guanidine as a solvent during hydroxylaminolysis. A low level of cleavage at Asn-X bonds has been observed in some cases, but aspartyl bonds appear to be resistant under conditions used. The infrequency of Asn-Gly bonds in most proteins results in the production of very large fragments that may overlap CNBr-produced fragments and could serve as new start points for sequential Edman degradation.
The amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V8 protease, Achromobacter protease I, and elastase) cleavages. Hydrazinolysis was also employed to determine the C-terminal amino acid. The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25,744. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively. The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases. Flavoxobin appears to be similar in secondary structure composition to batroxobin.
Several types of voltage-dependent calcium channels appear to occur in neurons, although coupling of the particular subtype of calcium channels to the release of neurotransmitter has not been clearly understood. We have examined the effects of subtype-specific inhibitors of the calcium channels on depolarization-induced release of endogenous neurotransmitters from brain slices. High potassium-induced release of glutamate and aspartate from hippocampal and striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from the hippocampal slices with almost the same potency as in the case of glutamate, whereas the potency in blocking the release of serotonin and dopamine from striatal slices was lower than that from the hippocampal slices. Another calcium channel blocker, omega-agatoxin TK, that was recently found to block P-type channels with very similar selectivity and potency to omega-agatoxin IVA, also inhibited the release of amino acid transmitters and monoamines, though its potency was lower than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA, partially inhibited the neurotransmitter release, but an L-type channel blocker, nifedipine was ineffective. We propose that the activation of P-type calcium channels makes a major contribution to depolarization-elicited neurotransmitter release in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and omega-agatoxin TK modulate the neurotransmitter release.