Tài liệu Báo cáo khoa học: Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity pdf

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 stru

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

1 Department of Microbiology, University of Virginia, Charlottesville, VA, USA

2 Laborato´rio Especial de Toxinologia Aplicada-CAT ⁄ CEPID, Instituto Butantan, Sao Paulo, Brazil

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

metallo-proteinase family of metallo-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 cate-gorized 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

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 4 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 How-ever, 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 signifi-cantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition Venom com-plexity, 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 com-plexity 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

Abbreviations

ER, endoplasmic reticulum; MMP, matrix metalloproteinase.

PIV class into the PIII class pending the finding of a

transcript in venom glands representing a PIV

struc-ture containing lectin-like domains In Fig 1, we show

a modified classification scheme that reflects the

nas-cent 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

com-plexity 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

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 func-tional 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 ven-oms Investigations on venom production in vivo have demonstrated that, like most secretory proteins, venom

Fig 1 Schematic of SVMP classes Question marks (?) in the figure indicate that the processed product has not been identified in the venom.

proteins are synthesized in the cytoplasm of secretory

cells in the gland and transferred to the rough

endo-plasmic 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

parti-cle on the endoplasmic reticulum (ER) During

trans-port 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,

glycosyla-tion, 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

pro-teins probably occurs, as with most latent protein forms

in the trans-Golgi network and nascent secretory

vesi-cles, 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 secre-tory 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 proteo-lysis 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 acti-vation, and could preclude wholesale degradation of the venom by activated SVMPs A hypothetical sche-matic of venom SVMP biosynthesis is presented in Fig 2, which typifies the protein biosynthetic pathway

of most eukaryotic cells

Fig 2 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.

Structural features of the SVMPs that

contribute to venom complexity

Role of disulfide bond patterns in

post-transla-tional 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

criti-cal 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

heat-shock proteins, protein disulfide isomerases and

pept-idyl-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 2 of [5])

However, the PI adamalysin contains five cysteinyl

res-idues, 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

sulfhy-dryl group in the protein, one would suspect that

disulfide bond shuffling could be promoted by the

resi-due; 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 do not appear to significantly affect the

crystal structures, in that all are very similar with

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, pro-teolytically 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 contrib-utes to the SVMP-derived venom complexity, and will

be further discussed below The proteinase domains are observed to have five, six or seven cysteinyl resi-dues, 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 pair-ing (Fig 3) This may explain why the proteinase domains, when processed from the nascent PII struc-ture, 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 pro-duct representing the disintegrin domain alone [30]

Fig 3 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].

Fig 3 Continued.

Fig 3 Continued.

Presumably, the post-translational processing

machin-ery in snake venom gland secretory cells must be

pre-disposed to this form of multimerization as compared

to dimerization between either disintegrin gene

prod-ucts 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

dis-integrin gene product and a PIIe SVMP This suggests

some structural or mechanistic advantage to having

one disintegrin domain residing in the structural

envi-ronment 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

atroly-sin 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

resi-dues 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

protein-ase 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,

catro-collastatin⁄ 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 observa-tion that the disulfide bond pattern of VAP2B⁄ catrocol-lastatin as observed from the crystal structure is rather different from that determined by MS for catrocollasta-tin 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 disinte-grin-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 iso-lated from viperid venoms [37,38], suggesting that it is not an isolated event As noted above, most PIII pro-teinase 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⁄ cyste-ine-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 cystei-nyls 374 and 390 (Fig 4; VAP1 numbering) Two pro-teins, 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 posi-tions 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

Fig 3 Continued.

cysteinyl residue in the PIIIbs jararhagin and

catrocol-lastatin This leads one to speculate that the presence

of an unpaired cysteinyl residue at that region of the

proteinase domain may be important for subsequent proteolytic processing of the disintegrin-like⁄ cysteine-rich domains from the PIII SVMP proteinase domain

Fig 4 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 5 Disulfide bonds of catrocollastatin ⁄ VAP2B disintegrin-like (underlined) and cysteine-rich (double-underlined) domains (AAC59672) revealed by the crystal struc-ture analysis (——) [33] and by N-terminal sequencing and MS ( ) [53] Cysteine resi-dues are numbered according to catrocol-lastatin ⁄ VAP2B [33] (- - - -) is the only coincident bond determined by both methods.

However, there must remain significant differences

between the processing of a disintegrin domain from a

nascent PIIa SVMP and that of the

disintegrin-like⁄ 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

Possi-ble explanations could be as simple as that the

disul-fide 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

pat-tern 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

obser-vation 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

pro-duce a disintegrin-like⁄ cysteine-rich domain

Interest-ingly, the N-terminus of the jararhagin C produced

in vitro was different from that observed in the

natu-rally occurring jararhagin C, suggesting several

possi-bilities 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

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 iso-meric form of jararhagin that is not normally pro-cessed 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 catrocollasta-tin C and those of the full-length catrocollastacatrocollasta-tin 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 catrocolla-statin 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 cyste-inyl pairs for catrocollastatin C (an explanation that

we feel to be unlikely) Another possibility is that during proteolytic processing of the nascent catrocol-lastatin, disulfide bond rearrangement occurred Alter-natively, the two different disulfide bond patterns representing folding isomers were in place before post-translational 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 pro-teins has been seen to be processed, there are folding isomers, perhaps promoted by the presence and rela-tive positions of the unpaired cysteinyl residues in the proteinase domain One of the folding isomers is read-ily processed to give rise to an unstable proteinase domain and a biologically active disintegrin-like⁄ cyste-ine-rich domain The other folding isomer is refractory