In order to optimize 2-DE separation of B. atrox venom proteins, linear and non-linear 3–10 pH gradients were tested in the isoelectric focusing (IEF) step. The non-linear gradient, developed to improve resolution of acidic proteins, provided better resolution of spots than linear pH gradient since many spots consisted of polypeptides displaying isoelectric points (pI) between 4 and 7 (data not shown). Two types of electrophoresis equipment – Multiphor II and IPGphor from GE Healthcare- were tested for the IEF step and both provided similar 2-DE maps (data not shown). On the other hand, for the second dimension, gradient gels (10–20% T) provided better 2-DE maps than 12% T gels, especially for proteins with molecular masses around 14 kDa (data not shown).
Patterns of protein spots visualized by silver staining were different between pooled venom samples from juvenile, sub-adult and adult B. atrox (Fig. 1). Their computer-assisted image analyses detected 110 spots in the gels from juveniles, 101 in sub-adults and 86 in adult venoms. Among the detected spots, 44 were found specifically in juveniles, 22 in sub-adults and 22 in adults. Image analysis also pinpointed substantial differences in the relative abundance of several spots matched at all three images (Table 1).
The identification of proteins was initially attempted by N-terminal sequencing of the proteins blotted to a PVDF membrane using Edman degradation. Because of the relatively low sensitivity of Edman degradation reliable peptide sequences were retrieved only from the most abundant spots. For instance, the group of seven spots of 23 kDa, more abundant in adults (group D, Fig.
1), presented the same N-terminal sequence (TPEQQRYVELLXVVD), where X stands for an undetermined amino acid. This sequence was 73 % identical to a fragment of the 23 kDa metalloproteinase bothrolysin [sptrembl: P20416] (1 TPEHQRYIELFLVVD 15) and to an internal sequence of the 50 kDa metalloproteinase bothrostatin [sptrembl: Q98SP2] (188 TPEHQRYIELFLVVD 202), both from
B. jararaca. However, Edman sequencing failed to identify a 52 kDa polypeptide that was detected as the most abundant spot in the 2-DE map of adult venom (group A, Fig.
1).
In-gel digests of non-identified spots were further analyzed by peptide mass fingerprinting (PMF). Only a few full length sequences of B. atrox proteins are currently available in a database, and therefore it is not surprising that PMF searches did not provide significant scores for any of the 2-DE spots present in the gels. However, this approach allowed us to group spots whose peptide mass fingerprints were similar. Based on the PMF data, all spots were arranged in 27 groups. The 2-DE maps of juveniles, sub-adults and adults showing the groups containing spots with similar PMF spectra are shown in Fig. 1. All groups were found in juvenile venom gels while adult gels displayed only 13 groups, revealing significant changes of B. atrox venom proteome during ontogenetic development.
One spot of each group was submitted to protein sequencing by tandem mass spectrometry followed by Mascot and MS BLAST database searches (Fig. 2), which enabled either to identify the protein, or to assign it to a class of highly homologous proteins. In this way, metalloproteinases, L-amino oxidases, serine proteinases, cysteine-rich secretory proteins (CRISPs), phospholipases A2, lectins and growth factors were identified (Table 1).
Given that very few sequences of B. atrox proteins are available in a sequence database, the best matches of MS BLAST database searches corresponded to proteins found in venoms of other snakes, mostly of the Bothrops genus (Table 1). Since several snake venom proteins share high sequence similarity and peptides analyzed by MS/MS cover only a small fraction of their sequences, it was not possible to unequivocally determine the protein homologues from the sequenced species. MS/MS analysis of the spot with apparent MW of 52 kDa (group A, Fig. 1 and Table 1) that was present in similar quantities in adult, sub-adult and juvenile gels is presented here as an example. MS/MS sequencing identified it as a member of the P-III class Zn-metalloproteinase. The best matches corresponded to the jararhagin and bothropasin, high molecular mass metalloproteinases from B. jararaca, which share approximately 95% of sequence identity. One of the determined sequences (KINPFR) is present in bothropasin, but not in jararhagin and another (BMYELANIVNEIFR) shares 100% identity with jararhagin only, because there is a substitution (F→ L) in bothropasin. The remaining peptide sequences (Table 1) are present in both bothropasin and jararhagin. Therefore, the 52 kDa spot is unequivocally related to a metalloproteinase from the P-III class that is homologous, albeit is different from both bothropasin and jararhagin.
The proteins from group C, whose molecular masses are similar to those of group A, were identified as metalloproteinases of the P-III class, albeit having higher pI values (7.1–7.5 comparing to pI 5 of group A). They share two peptide sequences (BKIPCAPEDVK and BGMVLPGTK) with the polypeptides from group A. Two other sequences (BXXVEVGEECDCGSPR and BLYCCVDSSPANK) matched bothropasin only partially and another sequence (BXXGTECQAA) occurs in metalloproteinases from other species of vipers. Differently from group A, the group C proteins are much more abundant in juveniles than adults as shown in Fig. 3.
Group D, one of the most prominent groups of spots in adult gels, comprised several isoforms of approximately 23 kDa and pI range between 5.3 and 6.4 (Fig 3). On the other hand, only two of these spots (in very low amounts) were detectable on silver stained gels of juvenile venom. Comparison of the three gels suggested that the concentration of 23 kDa isoforms increased during ontogenetic development (Fig. 3). N-terminal sequences of these proteins were determined by Edman degradation and the proteins were found to be homologous to bothrolysin and bothrostatin metalloproteinases (see above). MS BLAST searches with a query composed of peptide sequence proposals obtained by the de novo interpretation of MS/MS spectra produced as best hit a P-II class metalloproteinase from B. insularis. Snake venom metalloproteinases with molecular masses 23–25 kDa, which are composed by a sole metalloproteinase domain, are usually assigned to the P-I class [4,6]. Some P-I class proteins are produced by proteolytic processing of P-II metalloproteinases, which are larger and contain a desintegrin-like domain besides the metalloproteinase domain [7]. Therefore, proteolytic processing could explain the high similarity of proteins from group D with P-II class metalloproteinases. The spots from group D probably correspond to the 23 kDa polypeptide previously purified from B. atrox venom that constituted a single band in SDS-PAGE [2]. Further ESI-MS experiments suggested that there were at least three isoforms of this protein in the B. atrox venom [2]. Here, 2-DE gels were able to resolve at least seven isoforms in the venom of adults. Image analyses of the gels showed a remarkable increase in the volume of group D spots from juveniles to adults.
The group L comprised at least seven isoforms that were abundant in juvenile, less abundant in sub-adult and undetectable in adult venoms. They were homologous to berythractivase, a 78 kDa P-III class metalloproteinase found in B. erythromelas that is a protrombine activator that possess a high pro-coagulant activity [8].
The proteins from group M were also identified as metalloproteinases and were not detected in adults. These proteins were homologous to BOJUMET II from B. jararacusu as well as to berythractivase. Although the proteins from groups L and M were both homologous to berythractivase, PMF and MS/MS data indicated that they were, in fact, different proteins rather than post-translationally modified forms of the same gene.
Another group of spots (B) with molecular mass range of 55–61 kDa contained several isoforms present in similar amounts in the three different venom samples. Their sequences were homologous to apoxin I, a L-amino oxidase (LAO) from Crotalus adamanteus. LAO are major components of snake venoms that cause cell death by apoptosis [9].
The proteins from the groups E, N, Q and R were identified as serine proteinases. The snake venom serine proteinases possess thrombin-like activity and several of them have been isolated from bothropic venoms [4].
Proteins belonging to the class of Cysteine Rich Secretory Proteins (CRISP) were identified in group F and displayed similar expression in the three developmental stages analyzed. The CRISPs are found in epididimus and granular cells of mammals and seem to act in cell maturation of spermatozoa and cells from immune system, though the exact function of these proteins is unknown [10]. Members of CRISP family isolated from snake venoms, such as ablomin and trifilin, are responsible for blocking of smooth muscles contractions induced by depolarization. Immunological screening using anti-triflin antiserum identified CRISPs in different snake venoms, although the cross-reactivity was relatively low for the only member of the Bothrops (B. jararaca) genus tested [10].
In the low molecular mass range (12–15 kDa), several phospholipases A2 (PLA2) and C-type lectins were found. Within these groups, we identified at least four variant sequences of the same peptide stretch present in PLA2 (VAVLCFR, AAAVCFR, VAATCFR and VAVLYSR) indicating a high degree of polymorphism for these enzymes. Other examples of polymorphic peptide sequences were observed in groups W (more expressed in juveniles and sub-adults) and I (adult specific). MS BLAST searches produced as best hits the alpha chain of the C-type lectin bothrocetin, although the sequence of their peptides and the PMF analysis showed that they contained different proteins. C-type lectins were also identified as the major component in groups U and V. Interestingly, groups H (adults) and S (juveniles), despite having similar pI, molecular weight and some peptide sequences, actually contained different C-type lectins.
Proteins homologous to vascular endothelial growth factor (VEGF) from Bothrops insularis were detected in group X and their abundance decreased during ontogenetic development (Fig. 1). VEGFs from snake venoms are known to enhance the vascular permeability and play an important role in the initial stages of envenoming by Bothrops. It is assumed that snake venom VEGFs, dstimulate the distribution of the venom favouring both local and systemic actions [11].
Group Z displayed only one detectable spot that was present in juveniles and absent from adults. It was identified as a mixture of a nerve growth factor (NGF) and a C-type lectin. NGF belongs to a family of neurotrophic factors, which are endogenous soluble proteins regulating survival, growth morphological plasticity or synthesis of proteins for differentiated functions of neurons. In addition, there is increasing evidence that NGF activities are not restricted to the nervous system, but also affect non-neuronal cells, especially those of haematopoietic stem cell origin. NGF was previously described in the three main families of venomous snakes (Viperidae, Crotalidae and Elapidae). The ubiquitous presence of NGF in snake venoms suggests a toxinological importance of that protein in a sense of direct toxic action, indirect toxic action or an activity in the context of prey digestion [12].
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