Structural requirements of MLD-containing disintegrins for functional interaction with alpha 4 beta 1 and alpha 9 beta1 integrins

Three non-RGD-containing disintegrins, VLO5, EO5, and EC3, belong to the heterodimeric family of these snake venom-derived proteins. They are potent inhibitors of certain leukocyte integrins such as alpha4beta1, alpha4beta7, and alpha9beta1, and act through the MLD motif present in one of their subunits. However, the selectivity of these disintegrins to interact with integrins is related to the amino acid composition of the integrin-binding loop in the MLD-containing subunit. The most important amino acid is that preceding the MLD motif. In vitro experiments in adhesion and ELISA assays revealed that the TMLD-containing disintegrins, VLO5 and EO5, appeared to be very potent inhibitors of human alpha4beta1 and alpha9beta1 and less effective in inhibition of the alpha4beta7 integrin. The reverse effect was observed for the AMLD-containing disintegrin, EC3. The data with native disintegrins were confirmed by experiments with synthetic peptides displaying TMLD and AMLD motifs. The MLD-containing disintegrins showed differential activities to inhibit human and murine alpha4beta1 integrin. EC3 was a weaker inhibitor of human integrin, whereas VLO5 and EO5 less actively inhibited murine alpha4beta1. These data describe a useful set of potent and selective integrin antagonists and suggest conformational requirements of human and mouse integrins for interaction with ligands.     

Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin

KTS-disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of alpha1beta1 integrin. The amino acid sequence of the new KTS-disintegrin, viperistatin, differs from previously characterized obtustatin in three residues at position 24 (within the integrin binding loop), 38 (hydrophobic core) and 40 (C-terminal region). Noteworthy, viperistatin is about 25-fold more potent than obtustatin inhibiting the binding of this integrin to collagen IV. Synthetic peptides representing the full-length of integrin-binding loops of these disintegrins showed that the Leu24/Arg substitution appears to be partly responsible for the increased inhibitory activity of viperistatin over obtustatin.     

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.     

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.     

The presence of the WGD motif in CC8 heterodimeric disintegrin increases its inhibitory effect on alphaII(b)beta3, alpha(v)beta3, and alpha5beta1 integrins

Two highly homologous dimeric disintegrins, CC5 and CC8, have been isolated from the venom of the North African sand viper Cerastes cerastes. CC5 is a homodimer containing an RGD motif in its subunits. CC8 is a heterodimer. The CC8A and CC8B subunits contain RGD and WGD tripeptide sequence in their respective integrin-binding loops. Both CC5 and CC8 inhibited platelet aggregation and the adhesion of cells expressing integrins alphaII(b)beta3, alpha(v)beta3, and alpha5beta1 to appropriate ligands. However, the inhibitory activity of CC8 was at least 1 order of magnitude higher than that of CC5. Enhanced activity of CC8 over CC5 was also observed in the induction of LIBS epitopes on beta1 and beta3 integrins. Synthetic peptides in which the arginyl residue of the RGD motif had been replaced with tryptophans exhibited increased inhibitory activity toward integrins alpha5beta1, alphaII(b)beta3, and alpha(v)beta3. Moreover, alanine substitution of the aspartic acid of the WGD motif of these peptides decreased their inhibitory ability, whereas the same substitution in the RGD sequence almost completely abolished the activity of the peptides. We conclude that the WGD motif enhances the inhibitory activity of disintegrins toward alphaII(b)beta3, alpha(v)beta3, and alpha5beta1 integrins.     

Characterization of fertilin beta-disintegrin binding specificity in sperm-egg adhesion

An essential step leading to fertilization is the binding of a sperm to the egg plasma membrane. Fertilin beta, a membrane bound protein on the extracellular surface of sperm, partially mediates this binding via the alpha6beta1 integrin. Fertilin beta is a member of the still expanding family of ADAM proteins (a disintegrin and metalloprotease) that are implicated in many cellular functions ranging from neurogenesis to myoblast fusion and cytokine processing. Fertilin beta contains a highly conserved motif (D/E)ECD in the disintegrin domain. This suggests that (D/E)ECD could be the consensus sequence for recognition of disintegrins by alpha6beta1 integrins. Previously, it has been demonstrated that small peptides containing different moieties of this consensus sequence are inhibitors of in vitro fertilization. In the present study, we sought to determine whether a four amino acid peptide sequence with two adjacent acidic residues improved inhibition, and investigated the importance for inhibition of a cysteine versus a cystine. A series of linear and cyclic peptides were synthesized, in which either one or both adjacent acidic residues in the sequence DECD were mutated to their corresponding amides (N or Q). To explore the required oxidation state of the cysteine in the (D/E)ECD sequence, it was protected as a mixed disulfide. Our results indicate that only one acidic residue is required for inhibition of fertilization and a reduced C is required.     

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system

We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.     

Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin

The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.