Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

Disintegrin, hemorrhagic, and proteolytic activities of Mohave rattlesnake, Crotalus scutulatus scutulatus venoms lacking Mojave toxin

Venom from the Mohave rattlesnake, Crotalus scutulatus scutulatus, has been reported to be either: (1) neurotoxic; (2) hemorrhagic, or both (3) neurotoxic and hemorrhagic. In this study, 14 Mohave rattlesnakes from Arizona and Texas (USA) were analyzed for the presence of disintegrins and Mojave toxin. All venom samples were analyzed for the presence of hemorrhagic, proteolytic and disintegrin activities. The venoms were each chromatographed by reverse phase and their fractions tested for disintegrin activity. All specimens containing Mojave toxin were the most toxic and lacked proteolytic, hemorrhagic and disintegrin activities. In contrast, the venoms containing these activities lacked Mojave toxin. Two disintegrin genes, scutustatin and mojavestatin, were identified by PCR of genomic sequences. Scutustatin is a highly conserved disintegrin, while mojavestatin shows low conservation to other known disintegrins. Venoms with the highest LD50 measurements lacked both disintegrin genes, while the specimens with intermediate and low LD50 contained both genes. The intermediate LD50 group contained Mojave toxin and both disintegrin genes, but lacked hemorrhagic and disintegrin activity. Our results raise the possibility that scutustatin and mojavestatin are not expressed in the intermediate LD50 group, or that they may not be the same disintegrins responsible for the disintegrin activity found in the venom. Therefore, it is possible that Mohave rattlesnakes may produce more than two disintegrins.     

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein.     

Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis-inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis-inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino-acid residues. The amino-acid sequence predicted from the cDNA was highly homologous to VAP1. The amino-acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

Molecular Cloning of Echis ocellatus Disintegrins Reveals Non-Venom-Secreted Proteins and a Pathway for the Evolution of Ocellatusin

We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations. [Reviewing Editor: Dr. Bryan Grieg Fry]     

Molecular cloning and functional characterization of a snake venom metalloprotease.

A cDNA clone, MT-d, encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library. MT-d-I protein containing both metalloprotease and disintegrin domains, and MT-d-II protein containing the metalloprotease domain only were expressed in Escherichia coli and refolded successfully into their functional forms. Each of the refolded enzyme species exhibited distinct substrate specificity. Proteolytic activity of the MT-d-1 was able to hydrolyse type I gelatin, type-III and V collagens in contrast with the catalytic function of MT-d-II. MT-d-I protein having metalloprotease activity was also able to inhibit platelet aggregation. Functionally active MT-d-I protein underwent autoproteolytic processing in vitro to produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity. Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins.     

cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Crystal structure of the disintegrin heterodimer from saw-scaled viper (Echis carinatus) at 1.9 A resolution

Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 A resolution and belonged to space group P4(3)2(1)2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4(1)22 with similar cell dimensions. The structure refined to the final R(cryst)/R(free) factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42-Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them.     

Evolution of snake venom disintegrins by positive Darwinian selection

PII-disintegrins, cysteine-rich polypeptides broadly distributed in the venoms of geographically diverse species of vipers and rattlesnakes, antagonize the adhesive functions of beta(1) and beta(3) integrin receptors. PII-disintegrins evolved in Viperidae by neofunctionalization of disintegrin-like domains of duplicated PIII-snake venom hemorrhagic metalloproteinase (SVMP) genes recruited into the venom proteome before the radiation of the advanced snakes. Minimization of the gene (loss of introns and coding regions) and the protein structures (successive loss of disulfide bonds) underpins the postduplication divergence of disintegrins. However, little is known about the underlying genetic mechanisms that have generated the structural and functional diversity among disintegrins. Phylogenetic inference and maximum likelihood-based codon substitution approaches were used to analyze the evolution of the disintegrin family. The topology of the phylogenetic tree does not parallel that of the species tree. This incongruence is consistent with that expected for a multigene family undergoing a birth-and-death process in which the appearance and disappearance of loci are being driven by selection. Cysteine and buried residues appear to be under strong purifying selection due to their role in maintaining the active conformation of disintegrins. Divergence of disintegrins is strongly influenced by positive Darwinian selection causing accelerated rate of substitution in a substantial proportion of surface-exposed disintegrin residues. Global and lineage-specific sites evolving under diversifying selection were identified. Several sites are located within the integrin-binding loop and the C-terminal tail, two regions that form a conformational functional epitope. Arginine-glycine-aspartic acid (RGD) was inferred to represent the ancestral integrin-recognition motif, which emerged from the subgroup of PIII-SVMPs bearing the RDECD sequence. The most parsimonious nucleotide substitution model required for the emergence of all known disintegrin's integrin inhibitory motifs from an ancestral RGD sequence involves a minimum of three mutations. The adaptive advantage of the emergence of motifs targeting beta(1) integrins and the role of positively selected sites located within nonfunctional disintegrin regions appear to be difficult to rationalize in the context of a predator-prey arms race. Perhaps, this represents a consequence of the neofunctionalization potential of the disintegrin domain, a feature that may underlie its recruitment into the venom proteome followed by its successful transformation into a toxin.     

Disintegrin-like/Cysteine-Rich Region of ADAM 12 Is an Active Cell Adhesion Domain

ADAM (a disintegrin and metalloprotease) proteins contain structural homology to the P-III class of snake venom metalloproteases (SVMPs) and are postulated to function, by analogy to these SMVPs, as cell adhesion molecules. ADAM 12 has been implicated in fusion of myoblasts, but its mechanism of action is not known. Instead of the RGD-like cell-binding motif present in SVMP disintegrins, the disintegrin domain of ADAM 12 contains a unique SNS sequence and therefore its adhesive potential has been controversial. In this report we demonstrate that the disintegrin-like/cysteine-rich (DC) domain of ADAM 12 constitutes a functional cell adhesion domain. We have expressed the DC domain of mouse ADAM 12 in insect cells and shown that the recombinant protein supported adhesion of C2C12 myoblasts and NIH 3T3 fibroblasts in a divalent cation-dependent manner. A sulfhydryl-specific biotinylation reagent revealed, however, that the overall conformation and flexibility of the cell-binding region of ADAM 12 DC domain may be significantly different from those of the SVMP disintegrins. Moreover, the disulfide bond structure of the DC domain was critical for its function, as incubation of the recombinant protein with reducing agents abolished subsequent cell adhesion. Recombinant DC bound to C2C12 cells with high affinity (K(D) approximately 0.10 microM, total number of binding sites n approximately 4.6 x 10(5)/cell). Adhesive properties of the DC domain of ADAM 12 produced in insect cells were further confirmed by cell surface binding of the DC domain expressed in C2C12 cells and secreted to the medium, consistent with the role of ADAM 12 in cell-cell interactions and myoblast fusion.     

Amino acid sequence and homology modeling of obtustatin,a novel non-RGD-containing short disintegrin isolated from the venom of Vipera lebetina obtusa

Disintegrins represent a group of cysteine-rich peptides occurring in Crotalidae and Viperidae snake venoms, and are potent antagonists of several integrin receptors. A novel disintegrin, obtustatin, was isolated from the venom of the Vipera lebetina obtusa viper, and represents the first potent and selective inhibitor of the binding of integrin alpha(1)beta(1) to collagen IV. The primary structure of obtustatin contains 41 amino acids and is the shortest disintegrin described to date. Obtustatin shares the pattern of cysteines of other short disintegrins. However, in contrast to known short disintegrins, the integrin-binding loop of obtustatin is two residues shorter and does not express the classical RGD sequence. Using synthetic peptides, a KTS motif was identified as the integrin-binding sequence. A three-dimensional model of obtustatin, built by homology-modeling structure calculations using different templates and alignments, strongly indicates that the novel KTS motif may reside at the tip of a flexible loop.     

Isolation of bothrasperin, a disintegrin with potent platelet aggregation inhibitory activity, from the venom of the snake Bothrops asper

The venom of Bothrops asper induces severe coagulation disturbances in accidentally envenomed humans. However, only few studies have been conducted to identify components that interact with the hemostatic system in this venom. In the present work, we fractionated B. asper venom in order to investigate the possible presence of inhibitors of platelet aggregation. Using a combination of gel filtration, anion-exchange chromatography, and reverse-phase high performance liquid chromatography, we isolated an acidic protein which shows a single chain composition, with a molecular mass of approximately 8 kDa, estimated by SDS-polyacrylamide gel electrophoresis. Its N-terminal sequence has high similarity to disintegrins isolated from different snake venoms, which are known to bind to cellular integrins such as the GPIIb/IIIa fibrinogen receptor on platelets. The purified protein exerted potent aggregation inhibitory activity on ADP-stimulated human platelets in vitro, with an estimated IC50 of 50 nM. This biological activity, together with the biochemical characteristics observed, demonstrate that the protein isolated from B. asper venom is a disintegrin, hereby named "bothrasperin". This is the first disintegrin isolated from Central American viperid snake species.     

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.