Differences between Two Groups of Burmese Vipers (Viperidae: Azemiops) in the Proteomic Profiles, Immunoreactivity and Biochemical Functions of Their Venoms

Abstract

Two recently revised Azemiops snakes with apparent differences in their external appearances and skeletal morphologies but unclear genetic boundaries have been proposed. Some researchers have refrained from using the newly proposed taxonomy because these two "species" might be two clades corresponding to different geographical populations of Azemiops feae. To improve the understanding of the kinship of these two Burmese viper groups, more of their characteristics should be explored in depth. We performed a comparative analysis of the proteomic profiles and biochemical activities of snake venoms from these two groups (Sichuan A. feae and Zhejiang A. feae) and evaluated the immunorecognition capacity of commercial antivenoms toward them. Eight protein families were identified in venoms from these two groups, while phospholipase B was only detected in venom from Sichuan A. feae. These protein families displayed varying degrees of differences in relative abundance between venoms, and phospholipase A₂ (Sichuan A. feae: 57.15%; Zhejiang A. feae: 65.94%) was the predominated component. Gloydius brevicaudus antivenom exhibited the strongest capacity to immunologically recognize these two venoms, but this was mainly limited to components with high molecular masses, some of which differed between venoms. Additionally, Zhejiang A. feae venom was more toxic than Sichuan A. feae venom, and the venoms expressed remarkable differences in enzymatic activities, probably resulting from the variation in the relative abundance of specific protein families. Our findings unveil differences between the two Burmese viper groups in terms of proteomic profiles, immunoreactivity, and the biochemical functions of their venoms. This information will facilitate the management of snakebites caused by these snakes.

Keywords: Azemiops; biochemical activity; proteome; taxonomy; venom.

Figure 1

Sampling localities of the two Burmese vipers investigated in this study. (A) Sichuan Azemiops feae has a black head surface with a thin, yellow strip down the middle. (B) Zhejiang A. feae has a light head surface divided by two symmetrical dark stripes. The animal images were photographed by Jian-Fang Gao.

Figure 2

The venom proteomic profile of Sichuan A. feae. (A) Elution profile of venom proteins in RP-HPLC. The venom components were separated by a C18 column as described in the Materials and Methods section. (B) Electrophoretic profile of eluted fractions under reduced conditions. The protein bands were excised, tryptic digested, and analyzed by MALDI-TOF-MS/MS and nESI-MS/MS before being assigned to known protein families. CNP: C-type natriuretic peptide; CRISP, cysteine-rich secretory protein; HA: hyaluronidase; LAAO: l-amino acid oxidase; NGF: nerve growth factor; PLA₂, phospholipase A₂; PLB: phospholipase B; SVMP: snake venom metalloproteinase; SVSP: snake venom serine proteinase.

Figure 3

The venom proteomic profile of Zhejiang A. feae. (A) Elution profile of venom protein in RP-HPLC. The venom components were separated by a C18 column as described in the Materials and Methods section. (B) Electrophoretic profile of eluted fractions under reduced conditions. The protein bands were excised, tryptic digested, and analyzed by MALDI-TOF-MS/MS and nESI-MS/MS before being assigned to known protein families.

Figure 4

Comparison of simplified elution profiles in RP-HPLC between venoms from two A. feae groups. x-axis, retention time; y-axis, relative abundance of each chromatographical fraction in the total venom. The numbers (Sichuan A. feae/Zhejiang A. feae) at the end of the lines correspond to the fractions in the chromatography shown in Figure 2 and Figure 3 and indicate similar retention times and protein families for both venoms.

Figure 5

Relative abundance of venom toxin families in Sichuan A. feae (A) and Zhejiang A. feae (B). Unknown: unidentified components. The details of the identified venom proteins are listed in Supplemental Tables S1 and S2.

Figure 6

Cross-reaction between two Burmese viper venoms and four commercial antivenoms evaluated by ELISA. (A–D): commercial monovalent Deinagkistrodon acutus, Gloydius brevicaudus, Bungarus multicinctus and Naja atra antivenoms, respectively. The explanation for different lines in (B–D) is the same as that in (A). Data are expressed as the mean value ± SD (n = 3).

Figure 7

Cross-reaction between two Burmese viper venoms and commercial G. brevicaudus antivenom assessed by Western blotting. Left panel, SDS-PAGE profiles of venom protein (identified proteins were listed in Supplemental Tables S3 and S4); right panel, cross-reaction profiles of Western blotting; (A,C), Sichuan A. feae venom; (B,D), Zhejiang A. feae venom. The protein bands specifically expressed in both venoms are indicated by arrows, which on the left of lane A indicate the protein bands with molecular mass of ~27, 45, 66 and 73 kDa from bottom to top, and those on the right of lane B indicate ~59 and 75 kDa.

Figure 8

Myotoxicity of Sichuan A. feae and Zhejiang A. feae venoms in mice. Data are expressed as the mean value ± SD (n = 3).

Figure 9

Phospholipase A₂ activity of Sichuan A. feae and Zhejiang A. feae venoms determined using soybean lecithin. Data are expressed as the mean value ± SD (n = 3). It can be inferred that the substrate was greatly degraded by venoms at relatively high doses, and thus, the activity values of venoms appear to decrease significantly at 0.4 and 0.8 μg.