REVIEW ARTICLE Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity Jay W Fox1 and Solange M T Serrano2 Department of Microbiology, University of Virginia, Charlottesville, VA, USA ´ Laboratorio Especial de Toxinologia Aplicada-CAT ⁄ CEPID, Instituto Butantan, Sao Paulo, Brazil Keywords autolysis; disintegrin; disulfide bond; metalloproteinase; post-translational processing; proteome snake venom; structure; SVMP transcriptome Correspondence J W Fox, Department of Microbiology, University of Virginia, PO Box 800734, Charlottesville, VA 229080734, USA Fax: +1 434 982 2514 Tel: +1 434 924 0050 E-mail: jwf8x@virginia.edu (Received February 2008, revised 27 March 2008, accepted 15 April 2008) doi:10.1111/j.1742-4658.2008.06466.x As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree, the various sources of such complexity However, our knowledge is still far from complete The translation of genetic information from the snake genome to the transcriptome and ultimately the proteome is only beginning to be appreciated, and will require significantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition Venom complexity, however, is derived not only from the venom genomic structure but also from transcriptome generation and translation and, perhaps most importantly, post-translation modification of the nascent venom proteome In this review, we examine the snake venom metalloproteinases, some of the predominant components in viperid venoms, with regard to possible synthesis and post-translational mechanisms that contribute to venom complexity The aim of this review is to highlight the state of our knowledge on snake venom metalloproteinase post-translational processing and to suggest testable hypotheses regarding the cellular mechanisms associated with snake venom metalloproteinase complexity in venoms Introduction Since the first discovery of zinc-dependent proteinases in viperid snake venom, investigators have intensively studied the structure and function of these proteinases in order to understand their role in envenomation pathologies [1] With the advent of the first complete sequence determination of these proteinases, it was thought that they belonged to the matrix metalloproteinase family of proteinases [2] However, it soon became obvious that they in fact comprised a novel family of metalloproteinases, the M12 family, to which the ‘a disintegrin and metalloproteinase’ (ADAM) proteins also belong [3] As studies progressed, the snake venom metalloproteinases (SVMPs), as this group of proteinases is now named, were further categorized into the PI, PIIa and PIIb, PIIIa and PIIIb, and PIV classes [4,5] The criterion for this differential classification essentially was based on the presence or absence of various nonproteinase domains as observed via mRNA transcripts and proteins isolated in the venom To date, no PIV mRNA transcript has been observed, and thus it very well may be that the PIV structure simply represents another post-translational modification of the canonical PIII structure; therefore, in our new classification scheme, we have collapsed the Abbreviations ER, endoplasmic reticulum; MMP, matrix metalloproteinase 3016 FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano PIV class into the PIII class pending the finding of a transcript in venom glands representing a PIV structure containing lectin-like domains In Fig 1, we show a modified classification scheme that reflects the nascent P structural classes and well as the observed products following post-translational modification and processing Functionally, the SVMPs display a wide array of biological activities, many of which are toxic, and this scope of activities reflects the multitude of products derived from the four SVMP classes [5] Over the past several years, as the result of a variety of proteomic and transcriptomic investigations of snake venom and venom glands respectively, numerous databases have been generated that illuminate the complexity of snake venoms, particularly the viperid venoms Hundreds of proteins comprise viperid venom proteomes, and it is estimated that most viperid venoms are composed of at least 32% SVMPs, which suggests the potential for a significant role for the SVMPs in the pathologies associated with viperid evenomation [6–8] Given the apparent complexity of venoms and the SVMP components in venoms, one must question the molecular mechanisms responsible for this complexity [9] Superficially, one could simply Snake venom metalloproteinases and venom complexity attribute venom complexity to genomic ⁄ transcriptomic complexity, and to a certain degree this is the case However, over the past several years, there have been features, both structural and functional, reported in the literature, which suggest that there may be other factors involved in generating the observed complexity of the SVMPs in venom In this short review, we will discuss biosynthetic features of the SVMPs that we believe may be involved in SVMP structural and functional complexity, with the aim of generating renewed interest in understanding the molecular and cellular biology of the SVMPs Perhaps, as in the past, this review may also help guide us to the development of enhanced therapeutics for viperid snake envenomation and novel toxin-based drug discovery Venom protein biosynthesis Snake venoms are the products of specialized secretory glands located above the upper jawbone in venomous snakes These glands are considered to be specialized secretory glands evolved for the biosynthesis of venoms Investigations on venom production in vivo have demonstrated that, like most secretory proteins, venom Fig Schematic of SVMP classes Question marks (?) in the figure indicate that the processed product has not been identified in the venom FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3017 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano proteins are synthesized in the cytoplasm of secretory cells in the gland and transferred to the rough endoplasmic reticulum, and then the Golgi apparatus, and finally transported via secretory granules to the lumen of the venom gland [10] On the basis of cDNA studies, all venom proteins have a signal sequence that probably targets the nascent protein to a signal recognition particle on the endoplasmic reticulum (ER) During transport into the ER, the signal sequence is removed Presumably, like most secretory proteins, it is in the rough ER that the nascent venom proteins fold, and undergo disulfide bond oxidation ⁄ formation, glycosylation, and, in the specialized instances of some venom proteins, multimerization, such as is the case with dimeric disintegrins [PIId and PIIe, dimeric PIII class SVMPs (PIIIc)] and the multimeric PIIId class SVMPs (formerly PIV) As in other eukaryotic cells, in the venom gland secretory cells, incorrectly folded proteins would not be expected to enter into the Golgi network Proteolytic processing of the proforms of venom proteins probably occurs, as with most latent protein forms in the trans-Golgi network and nascent secretory vesicles, and is completed by the time that the vesicles are released into the venom gland lumen In only a few instances have sequences associated with the prodomain of SVMPs been detected in the venom [11] (Serrano, unpublished results), suggesting that, in fact, the bulk of the proteolytic processing of the proforms of the SVMPs occurs in advance of release of mature secretory vesicle contents into the lumen of the venom gland In the case of the SVMPs, activation may best occur in an environment in which widespread, random proteolysis of venom components is minimized Studies have suggested that the acidic pH of the venom gland lumen, in addition to pyrol-glutamate containing tripeptides, contributes to the lack of proteolytic activity of SVMPs in the gland [12,13] Likewise, the acidification of the secretory vesicles as they mature may give rise to an environment that could limit the proteolytic activity of processed SVMPs Therefore, the maturing secretory vesicle is probably the best environment for SVMP activation, and could preclude wholesale degradation of the venom by activated SVMPs A hypothetical schematic of venom SVMP biosynthesis is presented in Fig 2, which typifies the protein biosynthetic pathway of most eukaryotic cells Fig Schematic representing hypothetical biosynthetic pathways from transcription at the ER surface, through the endoplasmic reticulum to the Golgi, and release of the secretory vesicles into the venom lumen for the production of the three SVMP venom classes Parentheses in the figure indicate that the processed product has not been observed in the venom P, prodomain; M, metalloproteinase domain; S, spacer; D, disintegrin domain; DL, disintegrin-like domain; Cys, cysteine-rich domain; L, lectin-like 3018 FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano Structural features of the SVMPs that contribute to venom complexity Role of disulfide bond patterns in post-translational proteolytic processing of the SVMPs Venom proteins must maintain structural integrity in the oxidative extracellular world; hence, most have evolved to contain a number of disulfide bonds that stabilize the particular molecular scaffold that is critical for toxic action [14] as well as participate in the generation of multimeric venom proteins On the basis of studies of venoms using two-dimensional PAGE under reducing and nonreducing conditions, Serrano et al [15] clearly demonstrated that disulfide bond formation among venom proteins, leading to multimeric structures, has a profound effect on venom complexity Reducing gels of Crotalus atrox and Bothrops jararaca venom, as compared to nonreducing gels, indicated that apparent venom complexity is reduced by 60% due to disulfide bond crosslinking of venom proteins The biological synthesis of toxins, particularly the SVMPs, must entail a somewhat complex phenomenon that probably involves a variety of chaperones and other proteins to help ensure appropriate folding and disulfide bond pairing, as shown by the fact that heatshock proteins, protein disulfide isomerases and peptidyl-prolyl cis ⁄ trans isomerases have been identified in snake venoms and venom glands [6,16] The need for ancillary proteins for appropriate post-translational modifications is further substantiated by the fact that it has proven to be relatively difficult for investigators to produce recombinant SVMPs or, for that matter, their various domains in heterologous in vitro expression systems Cysteinyl residues in PI SVMPs range in number from four, to six, to seven, with two to three disulfide bonds being reported (Fig 3) (see also table of [5]) However, the PI adamalysin contains five cysteinyl residues, four of which are involved in disulfide bonds, leaving one free cysteinyl residue in the N-terminal region of the domain Given the presence of a sulfhydryl group in the protein, one would suspect that disulfide bond shuffling could be promoted by the residue; however, a consistent disulfide bond pattern is observed for specific venom proteins Most likely, incorrectly folded proteins and hence unusual disulfide bond patterns are removed from the post-translational process The crystal structures for eight PI SVMPs have been reported, and although there are different disulfide bond patterns observed among these PI SVMPs, they not appear to significantly affect the crystal structures, in that all are very similar with Snake venom metalloproteinases and venom complexity regard to secondary structure and folding [17–25] Thus, at least superficially, it seems that similar folding motifs and structures can give rise to different disulfide bonding patterns in the PI SVMPs Furthermore, there does not appear to be a correlation between disulfide bond pattern, folding and biological activity The PII SVMPs are distinguished by having a canonical disintegrin domain present in the nascent protein that, in most cases, is post-translationally, proteolytically processed from the proteinase domain It is important to note that the proteolytic products, the proteinase domain and the disintegrin domain, seem to be stable, in that they have been isolated from the crude venom [26,27] Thus, this represents one form of post-translational proteolytic processing that contributes to the SVMP-derived venom complexity, and will be further discussed below The proteinase domains are observed to have five, six or seven cysteinyl residues, most having three disulfide bonds paired in a similar way to that observed for the PI SVMPs As was the case with PI adamalysin, the unpaired, free cysteinyl residue, such as in the PIIa trigramin, is located in the N-terminal portion of the domain and does not seem to affect folding or disulfide bond pairing (Fig 3) This may explain why the proteinase domains, when processed from the nascent PII structure, are stable like the PI SVMPs An interesting exception to the typical PII SVMP processing scheme that gives rise to a proteinase and a disintegrin in venom is jerdonitin, a PIIb proteinase isolated from the venom of Trimeresurus jerdonii In jerdonitin, the processing of these domains does not appear to occur [28] The structural analysis of jerdonitin reveals two additional cysteinyl residues, one located in the spacer region and one in the disintegrin domain, which could promote a more compact structure that may preclude the proteolytic processing between the two domains (Fig 1) Here again, we observe that the generation of an additional SVMP structure based on variations in post-translational proteolytic processing contributes to SVMP-based venom complexity Some disintegrins, as processed from the nascent PII SVMPs, contain four, six (four intrachain and two interchain), six, seven or eight disulfide bonds [29] The predominance of disintegrins in venom in conjunction with the oxidation of monomeric disintegrins to form homodimeric and heterodimeric structures certainly contributes to the complexity of SVMP-based venom complexity Another interesting observation associated with the dimeric disintegrins is that it seems that many are formed by one subunit derived from a processed PII SVMP and another from a translated gene product representing the disintegrin domain alone [30] FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3019 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano Fig Sequence alignment of SVMPs (PI, green; PII, blue; PIII, red; PIIId, brown) by the program CLUSTALW Cysteine residues and putative N-glycosylation sites are highlighted in gray and green, respectively Disintegrin motifs (MGD; RGD; MVD; VGD; KGD) are shown in red The hypervariable region is highlighted in yellow Cysteine residues are numbered according to the VAP1 sequence [32] 3020 FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano Snake venom metalloproteinases and venom complexity Fig Continued FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3021 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano Fig Continued 3022 FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano Snake venom metalloproteinases and venom complexity Fig Continued Presumably, the post-translational processing machinery in snake venom gland secretory cells must be predisposed to this form of multimerization as compared to dimerization between either disintegrin gene products or between two disintegrins processed from the PIId structure As mentioned, disulfide bond formation typically occurs prior to proteolytic processing in the trans-Golgi or secretory vesicles Thus, if disulfide bond pairing to form a heterodimeric disintegrin occurs, it presumably happens between the nascent disintegrin gene product and a PIIe SVMP This suggests some structural or mechanistic advantage to having one disintegrin domain residing in the structural environment of the unprocessed PIIe for the formation of the heterodimer later in the post-translational process The PIII SVMPs are the most intriguing of the SVMP categories in terms of their contribution to venom complexity and function One of the first sequences determined for a PIII SVMP was atrolysin A from C atrox The mature protein was observed to be composed of three domains: a metalloproteinase domain, a disintegrin-like domain, and a cysteine-rich domain [31] The metalloproteinase domains of the PIII SVMP class have either six or seven cysteinyl residues with three disulfide bonds The disulfide bond pattern of the PIII proteinase domains appears to be different from that observed in the PI or PII proteinase domains (Fig 4) Most of the PIIIs have an odd (seventh) cysteinyl residue in the proteinase domain The positions of the seventh cysteinyl residues in the PIIIs appear to segregate to either the region near the cysteinyl cluster of residues 350, 352 and 357 or between cysteinyl residues 374 and 390 (Fig 4) Recently, crystal structures for three PIII SVMPs have been determined: VAP1, a dimeric PIIIc, catrocollastatin ⁄ VAP2B, a PIIIb, both from the venom of C atrox, and RVV-X, a PIIId from Vipera russelli [32–34] Many important observations regarding PIII structure and function were obtained from those studies The first is that although the disulfide bond patterns are different in these PIIIs from that observed in the PI SVMPs, the crystal structures of the metallo- proteinase domains are very similar This underscores the point that similarly folded domains can support different disulfide bond patterns Second, and perhaps most interesting, is the observation that the disulfide bond pattern of VAP2B ⁄ catrocollastatin as observed from the crystal structure is rather different from that determined by MS for catrocollastatin C, the processed disintegrin-like and cysteine-rich domain from catrocollastatin (Fig 5) This observation will be further discussed in the next section In 1994, Usami et al isolated from the venom of B jararaca a protein that was composed of a disintegrin-like and a cysteine-rich domain [35] Sequence analysis of the protein indicated that it was the processed disintegrin-like and cysteine-rich domains from the PIIIb hemorrhagic toxin jararhagin [36] Since then, several other processed disintegrin-like and cysteine-rich domains from PIII SVMPs have been isolated from viperid venoms [37,38], suggesting that it is not an isolated event As noted above, most PIII proteinase domains have an unpaired cysteinyl residue Upon examination of the proteinase domains of two of the PIIIbs that appear to undergo post-translational proteolytic processing to yield a disintegrin-like ⁄ cysteine-rich domain product in the venom, there does seem to be some structural similarity The unpaired cysteinyl residues of both jararhagin and catrocollastatin are located at position 379 in the loop between cysteinyls 374 and 390 (Fig 4; VAP1 numbering) Two proteins, which to date have not been observed to be processed in the venom gland, atrolysin A and VAP1, have their unpaired cysteinyl residues located at positions 360 and 365 respectively, N-terminal to the unpaired cysteinyl residues observed in jararhagin and catrocollastatin Jarahagin and catrocollastatin are known to undergo post-translational processing to yield a disintegrin-like ⁄ cysteine-rich domain product in the venom Furthermore, it is interesting to note that the PII atrolysin E, which is readily processed to yield a stable proteinase domain and free disintegrin in the venom, also has an unpaired cysteinyl residue located at position 379, the same position as the unpaired FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3023 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano Fig Sequence alignment of SVMP catalytic domains Disulfide bonds of adamalysin II ( ) [17] and VAP1 (—) [32] revealed by crystal structure analysis are depicted Cyteine residues are numbered according to VAP1 [32] Cysteine residues not involved in bonding in adamalysin II and catrocollastatin ⁄ VAP2B are shown in bold; Cys365 of VAP1 is underlined Fig Disulfide bonds of catrocollastatin ⁄ VAP2B disintegrin-like (underlined) and cysteine-rich (double-underlined) domains (AAC59672) revealed by the crystal structure analysis (——) [33] and by N-terminal sequencing and MS ( ) [53] Cysteine residues are numbered according to catrocollastatin ⁄ VAP2B [33] (- - - -) is the only coincident bond determined by both methods cysteinyl residue in the PIIIbs jararhagin and catrocollastatin This leads one to speculate that the presence of an unpaired cysteinyl residue at that region of the 3024 proteinase domain may be important for subsequent proteolytic processing of the disintegrin-like ⁄ cysteinerich domains from the PIII SVMP proteinase domain FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano However, there must remain significant differences between the processing of a disintegrin domain from a nascent PIIa SVMP and that of the disintegrinlike ⁄ cysteine-rich domain from a PIIIb SVMP To date, it seems that the processed proteinase domains from PIIas are stable and can be isolated from the venom [39], whereas no processed proteinase domain from a PIIIb has been isolated from the venom Possible explanations could be as simple as that the disulfide bond patterns found in PIIa SVMPs allow for a stable proteinase domain when not in the presence of the disintegrin domain, whereas the disulfide bond pattern and the odd cysteinyl residue in the PIIIb domains are such that the domain is unstable and perhaps susceptible to degradation by the numerous proteinases in the venom For example, isolated PIIIbs such as bothropasin and brevilysin H6 can be induced to undergo autolysis in vitro, when the proteinase domain is observed to be degraded after release of disintegrin-like ⁄ cysteine-rich (DC) domains [40,41] Another possible reason for these differences, although perhaps less likely, is that the cellular mechanisms of post-translation proteolytic processing of the PIIas and PIIIbs are different Several years ago, we became intrigued by the observation that both the processed and unprocessed forms of PIIIs could be found in venoms For example, both full-length jararhagin and catrocollastatin and their processed disintegrin-like ⁄ cysteine-rich domains (jararhagin C and catrocollastatin C) products could be isolated from their respective crude venoms, but not the product proteinase domain [37,42] This led us to ask the question as to why both forms were found in the venom; why are not 100% of those particular PIIIb SVMPs completely processed rather than only a fraction of them? We proceeded to investigate this by attempting to manipulate jararhagin in vitro to undergo autolysis What we observed during the course of those experiments was the following: (a) under most conditions, jararhagin as isolated from the venom is stable against autolysis, as would be expected, given that it is present in the venom; and (b) only under conditions such alkaline pH, low calcium or the presence of reducing agents did some low fraction of jararhagin undergo autolysis to produce a disintegrin-like ⁄ cysteine-rich domain Interestingly, the N-terminus of the jararhagin C produced in vitro was different from that observed in the naturally occurring jararhagin C, suggesting several possibilities Perhaps the jararhagin C found in venom may not be an autolysis product, but rather a product from a different proteinase, or perhaps the structure of the jararhagin that is processed in venom is different from Snake venom metalloproteinases and venom complexity that of the jararhagin that is not processed We feel that the latter scenario is more likely to be correct, because when the jararhagin is artificially perturbed in vitro to undergo autolysis, the alternative site observed for the proteolysis is due to a structural isomeric form of jararhagin that is not normally processed during biosynthesis Several conclusions and ⁄ or questions result from these experiments From a single venom pool, we have isolated 3% of full-length jararhagin and 0.5% of jararhagin C [42] Thus, from this example, we can estimate that approximately only one-quarter of the jararhagin synthesized is processed to jararhagin C The question is why only 25% of the jararhagin synthesized is processed to jararhagin C The jararhagin found in the venom is relatively stable against processing in vitro, and this suggests to us the possibility that there are multiple isoforms of nascent jararhagin, such as might be the case with folding isomers, only a limited number of which are susceptible to processing to the end-product of jararhagin C As seen in Fig 5, the disulfide bond patterns of processed catrocollastatin C and those of the full-length catrocollastatin are different One disulfide bond pairing is shared between the two structures Furthermore, as seen in Fig 5, close inspection of the disulfide bond pairing in the disintegrin-like domain of VAP2b indicates that the disulfide bond pattern determined for catrocollastatin C could not occur without significant structural rearrangement ⁄ folding The different disulfide bond patterns observed between VAP2b ⁄ catrocollastatin and catrocollastatin C may be explained by disulfide bond shuffling during experimental determination of cysteinyl pairs for catrocollastatin C (an explanation that we feel to be unlikely) Another possibility is that during proteolytic processing of the nascent catrocollastatin, disulfide bond rearrangement occurred Alternatively, the two different disulfide bond patterns representing folding isomers were in place before posttranslational proteolytic processing and only one of those isomeric forms (the catrocollastatin C form) was processed We hypothesize that it is most likely that the former case is valid In summary, we propose the following scenario for PIIIb post-translational proteolytic processing: In the case of catrocollastatin and jararhagin, where a certain population of the proteins has been seen to be processed, there are folding isomers, perhaps promoted by the presence and relative positions of the unpaired cysteinyl residues in the proteinase domain One of the folding isomers is readily processed to give rise to an unstable proteinase domain and a biologically active disintegrin-like ⁄ cysteine-rich domain The other folding isomer is refractory FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3025 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano to proteolytic processing in the ER ⁄ Golgi system, and is found in the venom in the form of the canonical PIIIa three-domain structure As cDNA sequences for various P classes of the SVMPs became available, it was clear that there was a short sequence coding for approximately 10 residues or so immediately C-terminal to the proteinase domain, which in the case of the PII and PIII classes was followed by the disintegrin-like domain [31] We termed this region the ‘spacer’, suggesting that it provides a structural space between the proteinases and disintegrin or disintegrin-like domains that could be involved in post-translational modification of the nascent SVMPs As shown in Fig 1, the SVMPs found in the venom demonstrate a variety of differences with regard to the presence of spacer sequences in the mature proteins For example, in the case of the PIa SVMP atrolysin C, the spacer domain is processed from the proteinase during post-translational modification, whereas in the PIIa atrolysin E, the spacer sequence is still found C-terminal to the proteinase For the processed PIIIb catrocollastatin C, there is a spacer sequence N-terminal to the disintegrin-like domain Thus it appears that for some members in each of the P classes, a degree of proteolytic processing occurs at the spacer (Fig 1; PIa, PIIa, PIId, PIIe, PIIIb) This suggests a functional role for the spacer domain in providing accessible proteolytic processing sites to give those structural classes, whereas in some PIII SVMPs and long distintegrins, the spacer region is not processed, and as such is an integral part of those structures From the crystal structures of the monomeric SVMP catrocollastatin ⁄ VAP2b, the spacer region, or linker as per their terminology, appears to be accessible to the solvent and contributes to the flexibility observed between the proteinases and the disintegrinlike domains [33] However, in the case of this specific molecule, processing did not occur, suggesting that simple solvent accessibility of the spacer is not sufficient for processing Overall, spacer accessibility is probably associated with the structure of the adjacent proteinase and disintegrin-like domains and the packing of those two domains together in the structure Comparison of the sequences in the spacer domain among the P classes shows significant sequence conservation and leads to no obvious conclusions as to why some spacer regions are cleaved and some are not, based on simple peptide bond specificities of processing proteinases Thus, at this point we can only suggest that the overall accessibility of the spacer to processing enzymes during post-translational processing is necessary, but not sufficient, for proteolytic processing 3026 The PII SVMP class precursor gives rise to a minimum of five product classes in the venom (Fig 1) In fact, this precursor class is probably responsible for a significant amount of the venom complexity associated with the SVMPs, in that the PII precursor class gives rise to a proteinase and a disintegrin As is observed with the PII SVMP class, the PIII class also significantly contributes to venom complexity In Fig 1, it can be seen that there are essentially four protein product classes found in the venom from the PIII precursor Thus, the PIII class is also one of the more prolific in terms of contributing to both the structural and functional complexity of the venom Interesting structural features in disintegrin, disintegrin-like and cysteine-rich domains There are several interesting structural features associated with SVMP disintegrin, disintegrin-like and cysteine-rich domains that warrant some discussion The most obvious and interesting structural difference between the disintegrin-like domain of the PIII class and the disintegrin domain of the PII class is the presence of a cysteinyl residue in the PIII disintegrin-like domain, residing in what is called the RGD loop in disintegrin domains (Fig 6; residue 468, VAP1 numbering) The potent integrin-binding inhibitor activity associated with the disintegrins was proposed to be the evolutionary result of the loss of the cysteinyl residue in this region, thereby structurally freeing the loop for integrin interaction, coupled with the presence of the integrin-binding RGD motif [29] Other structural alterations probably occurred during the evolution of the disintegrin domains, expanding their repertoire of possibilities to interact with integrins but without causing drastic fold changes Indeed, disintegrin domains show similar overall structures, although there appear to be as many as three different disulfide bond patterns in the disintegrins [43] As seen in Fig 6, four disulfide bond pairs located in the center and in the C-terminal region of the domains are shared between the disintegrin flavoridin and VAP1 The disulfide bond patterns of the disintegrins and disintegrin-like domains are different in the N-terminal region, and reflect the fact that the lack of the first cysteinyl residue (Fig 6; residue 406, VAP1 numbering) in the disintegrins gives rise to the necessity for a different disulfide bond pattern in this region The C-terminal region of the PIII class has been termed ‘cysteine-rich’, due to the abundance of cysteinyl residues (13 residues) in this domain Interestingly, neither the disintegrin-like nor the cysteine-rich domain from the PIII class has been separately FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS J W Fox and S M T Serrano Snake venom metalloproteinases and venom complexity Fig Sequence alignment of disintegrins (blue) trigramin (CAA35910), flavoridin (1FVLA), contortrostatin (Q9IAB0) and bitistatin (P17497), and disintegrin-like (blue) ⁄ cysteine-rich domains (red) of the SVMPs atrolysin A (AAA03326), jararhagin (CAA48323), VAP1 (2EROA), BjussuMP_I (ABD73129) and kaouthiagin (P82942)] by the program CLUSTALW Cysteine residues are highlighted in gray Disintegrin and disintegrin-like sequences are shown in italic bold type Disulfide bonds of VAP1 [32] and flavoridin [54,55] are depicted (———) Common disulfide bonds between VAP1 and flavoridin are shown in red Cysteine residues are numbered according to the VAP1 sequence [32] isolated from venom; they have only been found as a single biologically active protein composed of these domains [35,37] This suggests that, structurally, these domains are very closely associated, which may preclude further processing Over the past several years, evidence has been mounting which indicates that the functionality attributed to the disintegrin-like ⁄ cysteine-rich proteins found in venom resides in the cysteine-rich domain region [44–49] The biological activities attributed to this domain result from its ability to interact with other proteins, such as FACIT collagens, von Willebrand factor and integrins Furthermore, in the case of the PIIId factor X activating proteinase, RVV-X (formerly classified as a PIV class member), the hypervariable region in the cysteine-rich domain has been suggested to manifest the functionality (factor X activation) of the protein by its ability to interact with the first lectin-like subunit of the multimeric structure via a unique cysteinyl residue found in the hypervariable region [34] Finally, there are three PIII class SVMPs, HR1B, from the venom of Trimeresurus flavoviridis [50], BjussuMP_I, from Bothrops jararacussu [51], and kaouthiagin, from Naja kaouthia [52], that have the integrin-binding motifs KGD or RGD in their cysteine-rich domains (Fig 6) Comparing the sequences of these SVMPs with those of other PIII SVMPs, one can observe, in the case of BjussuMP_I, that cysteinyl residues at positions 406, 425 and 468 (VAP1 numbering) are absent from the disintegrin-like domain In kaouthiagin, the disintegrin-like domain lacks cysteinyl residues at positions 443, 448, 449 and 452 (VAP1 numbering) This might reflect a different folding structure in the N-terminal regions of the disintegrin- like domains of these two proteins, although the C-terminal regions of the domains are probably similar to those of the rest of the class Whether the different disulfide bond patterns of these two proteinases are in some way structurally related to the presence of the integrin-binding motifs in their cysteine-rich domains and somehow impact on the function of the proteins is unknown; ultimately, additional functional studies and crystal structures will be required to fully appreciate the relevance of these structures in the context of other PIII SVMPs Concluding remarks In this review, we have attempted to present, based on the literature, the current understanding of the structural features that contribute to the complexity observed in many snake venoms The significance of this resides in the concept that the pathophysiological effects observed following envenomation can be attributed to the combination of individual activities of discrete toxins in the venom as well as, in some cases, synergistic effects among the toxins, and therefore understanding venom complexity and the source of such complexity represents an important challenge in the field Although recent transcriptome and proteome studies on venoms have provided us with tremendous insights into venom complexity, very little is empirically known about how the biosynthetic process of nascent venom protein production contributes to the complexity It is our hope that this review will provide a solid foundation on which future investigations can be conducted to illuminate the biosynthetic processes that give rise to venom complexity and, as such, FEBS Journal 275 (2008) 3016–3030 ª 2008 The Authors Journal compilation ª 2008 FEBS 3027 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano lead to a better understanding of venom action and possibly therapeutic intervention 13 References Takahashi T & Osaka A (1970) Purification and some properties of two hemorrhagic principles (HR2a and HR2b) in the venom of Trimeresurus 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Journal compilation ª 2008 FEBS ... [9] Superficially, one could simply Snake venom metalloproteinases and venom complexity attribute venom complexity to genomic ⁄ transcriptomic complexity, and to a certain degree this is the case... 2008 FEBS 3017 Snake venom metalloproteinases and venom complexity J W Fox and S M T Serrano proteins are synthesized in the cytoplasm of secretory cells in the gland and transferred to the rough... variety of proteomic and transcriptomic investigations of snake venom and venom glands respectively, numerous databases have been generated that illuminate the complexity of snake venoms, particularly