Molecular isoforms of cobra venom factor-like proteins in the venom of Austrelaps superbus.
Cobra venom factor (CVF) is characteristic of the elapid cobras and has not been reported from venoms of any other families of snakes. During our search for novel proteins, we isolated a polypeptide from the venom of the snake Austrelaps superbus (Lowland Copperhead) that showed structural similarity to C-terminal segment of the alpha-chain of CVF and hence named as AVFalphac (AVF-A. superbus venom factor). cDNA sequence of AVFalphac and its precursor indicated the presence of two isoforms of CVF-like proteins in A. superbus venom gland. This is the first report of molecular isoforms of CVF-like proteins in the venom of an Australian elapid snake. We have determined the complete cDNA sequence of both the isoforms (AVF-1 and AVF-2). They differ in their potential glycosylation sites and the characteristic thioester bond sequence. They display the overall domain structure of CVF and complement C3 proteins. By real-time quantitative analysis, we show that there is a 140-fold difference in the mRNA expression levels of the two isoforms in the venom gland of A. superbus. We also show the presence of AVF-1 and its variant (not AVF-2) in A. superbus venom by partial purification, dot blots, Western blots and peptide mapping using mass spectrometry. Partially purified proteins activate human Factor B in the presence of Factor D and Mg(2+), and deplete the complement activity in human and guinea pig serum. The bimolecular complex (AVFBb) formed activates complement C3 but not complement C5. Thus, AVF proteins may serve as potential candidates for therapeutic complement depletion without side effects. Thus, the discovery of CVF-like proteins in the venom of this Australian elapid snake provides an alternative source of research tools, and contributes to our understanding of the structure-function relationships and evolution of new members of CVF-like proteins.
Rehana
Syed
S
Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543, Singapore.
Manjunatha Kini
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D002851:Chromatography, High Pressure Liquid; D003176:Complement C3 / Q000737:chemistry; D018076:DNA, Complementary; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017815:Elapidae / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003176:Complement C3; D018076:DNA, Complementary; D004546:Elapid Venoms; D010455:Peptides; D020033:Protein Isoforms; C016310:cobra venom factor