Snake venomics of Bitis gabonica gabonica.: Protein family composition, subunit organization of venom toxins, and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2

The protein composition of the venom of the East African Gaboon viper (Bitis gabonica gabonica) was analyzed using RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting, and CID-MS/MS. In total, 35 proteins of molecular masses in the range of 7-160 kDa and belonging to 12 toxin families were identified. The most abundant proteins were serine proteinases (26.4%), Zn2+-metalloproteinases (22.9%), C-type lectin-like proteins (14.3%), PLA(2)s (11.4%), and bitiscystatin (9.8%). Other protein classes, that is, bradykinin-potentiating peptides, dimeric disintegrins, Kunitz-type inhibitor, DC-fragments, sv-VEGF, CRISP, and L-amino acid oxidase, comprised between 1.3 and 3.4% of the total venom proteome. Only 11 venom-secreted proteins matched any of the previously reported 22 partial or full-length venom gland transcripts. In addition, venome and transcriptome depart in their relative abundances of different toxin families. The proteomic characterization of purified B. gabonica gabonica proteins run under nonreducing and reducing SDS-PAGE conditions revealed their aggregation state and subunit composition. Multimeric proteins included heterodimeric disintegrins, homodimeric sv-VEGF-A, heterodimeric (alpha beta) and tetrameric (alpha beta)(4) C-type lectins, and multimeric PIII Zn2+-metalloproteinases. Determination of the complete primary structure and subunit composition of the two major dimeric disintegrins, bitisgabonin-1 and bitisgabonin-2, showed that each comprised a distinct RGD- and MLD-bearing subunit and a common, N-terminal-blocked, RGD-containing subunit identical to the disintegrin domain of the PII Zn2+-metalloproteinase 4. Cell adhesion inhibition assays showed that bitisgabonin-1 (RGD-RGD) is a potent inhibitor of integrin alpha(5)beta(1), whereas bitisgabonin-2 (MLD-RGD) is a better antagonist of integrins alpha(4)beta(1) and alpha(9)beta(1).