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Snake venom is a complex mixture of proteins and peptides, and a …
Biology Articles » Zoology » Herpetology » Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys » Results
According to the classification described above, the distributions of the identified proteins in five categories are listed in Table 1. As expected, Naja venom contains high amounts of cardio- and neuro-toxins, both occupying approx. 60% of the composition, and Agki has a high abundance of proteins involved in disruption of the haemostatic system, with 28% of haemotoxins and 40% of metalloproteinases. The content of phospholipase A2 in both venoms is close, at 18% and 14% respectively. Although some identified proteins have diverse amino acid sequences, these proteins may belong to the same protein family, at least performing similar biological functions. For instance, five unique proteins identified in Naja by this approach are assigned as GI numbers 1000502, 1326087, 1661022, 1134871 and 299268 that are the members of cardiotoxin 1 family with over 80% identity of amino acid sequence (see Supplementary Tables at http://www.BiochemJ.org/bj/384/bj3840119add.htm).
These identified venomous proteins or peptides are categorized in Table 1. In Naja venom, 74% of the identified proteins and peptides are cardiotoxins and neurotoxins, but only one haemotoxin was found in this venom. In contrast with Naja, Agki venom has 23% haemotoxin and 30% metalloproteinases, but only three cardiotoxins were detected, in which cardiotoxin-2c (GI number 3342766) is commonly found in both venoms. Comparing the differences between theoretical and apparent values of MM, 94% of the proteins identified from Naja venom display good correlations, whereas 46% of Agki proteins closely matched their theoretical predictions. Over 50% of Agki proteins migrated on SDS/PAGE with significantly low apparent MMs, suggesting that the degradation caused by proteases is common in Agki venom. Interestingly, none of the phospholipases A2 in Agki were found degraded; on the contrary, more than 80% of metalloproteinases appeared as the reduced molecular sizes on SDS/PAGE.
The fractions containing proteins with MMNaja and Agki venom respectively. Of these spectra, approx. 10% gave reliable candidate peptides by searching the snake venom protein database. Totals of 77 and 48 unique proteins or peptides were confirmed for Naja and Agki venom respectively (Table 1; details in Supplementary Table I at http://www.BiochemJ.org/bj/384/bj3840119add.htm). The proteins with apparent sizes more than 10 kDa were analysed by 2DE and subjected to MALDI–TOF-MS after staining by Coomassie Brilliant Blue and in-gel digestion as described above. A mass fingerprint of the unfractionated peptide mixture was obtained by MALDI–TOF-MS followed by Mascot protein identification mapping. To ensure the accuracy of protein identification, the following criteria were followed for each sample: (i) ranked in top two hits; (ii) at least four matched sequences; and (iii) over 10% protein sequence coverage. Totals of 190 and 169 spots from Naja and Agki respectively were picked from the corresponding gel. For Naja venom, 152 high-quality MALDI–TOF-MS spectra were collected and 100 spectra were matched to the correct peptides. From these samples, 16 unique proteins were assigned. For Agki venom, 133 high-quality MALDI–TOF-MS spectra were collected and 85 spectra matched the correct peptides. Agki venom had 15 unique confirmed proteins (Table 1; details in Supplementary Table II at http://www.BiochemJ.org/bj/384/bj3840119add.htm).
Following Sephadex G-50 GF, the pool of small venom proteins is expected to contain few, if any, large proteins (MM>10 kDa). Peptide identification of Naja venom by LC-MS/MS indicated that approx. 90% of proteins were cardio- and neuro-toxins, and over 91% had a MM less than 10 kDa. Protein degradation was apparent in the pool collected from late fractions of GF from the Agki sample, where approx. 90% of the proteins identified by MS have the mass values of more than 10 kDa. Surprisingly, the number of unique proteins verified by MALDI–TOF-MS from 2DE gel spots was much less than that of the identified proteins, with an average of six spots/unique protein. These data suggest that degradation or modification were prevalent in both samples. For instance, in Naja venom, 5′-END (5′-ectonucleotidase) from Rattus norvegicus (GI number 11024643) has theoretical pI and MM values of 6.5 and 64 kDa respectively. A total of 28 spots from 2DE were verified as 5′-END, with MMs ranging from 29 to 64 kDa and pIs ranging from 4.9 to 9.0. In Agki venom, a metalloproteinase from Gloydius halys (GI number 4106001) has theoretical pI and MM values of 5.3 and 70 kDa respectively; however, eight protein spots were identified as this enzyme on 2DE, with MMs ranging from 28 to 62 kDa and with pI values in the narrow range of 4.5 to 5.3. Interestingly, several venomous proteins were found to have higher MMs than their theoretical values. Haemorrhagic metalloproteinase kaouthiagin (GI number 32469675; 44 kDa) in Naja and salmobin (GI number 3668352; 29 kDa) in Agki migrated with MMs of 100 and 48 kDa respectively.
As mentioned above, snake venom proteins are broadly categorized into three groups based on toxicity and two groups based on sequence homology. A general summary of the proteomes of Naja and Agki venom is briefly discussed below.
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