Meeting Abstract
Previous transcriptomic analysis of the venom gland of the king cobra (Ophiophagus hannah) yielded a gene for a protein similar to neprilysin, along with many other toxin genes. Although all known venom proteins are secreted into the venom, this gene did not contain a signal peptide, indicating that it is not secreted. Rather, it contained a transmembrane domain, which indicates that it is an integral membrane protein. However, proteomic analysis indicated the presence of this protein in the venom of the same animal. We isolated and purified this protein using size exclusion and anion exchange chromatography and verified its presence through tandem mass spectrometry of peptides obtained by digestion with trypsin and Glu-C. Overall, we were able to obtain greater than 75% coverage of the sequence of the protein, and this coverage included portions N- and C-terminal to the transmembrane domain. We further utilized the protein at various stages of purification for assays against atrial natriuretic peptide, a natural substrate of neprilysin. Intraspecific genomic and transcriptomic sequence analysis showed the sequences to be identical, and exon comparisons with other genomic sequences showed strong similarities. Overall, these data indicate that the protein found in the venom is identical to that found in other tissues, and that the protein is neprilysin. This represents the first description of a membrane-bound protein isolated from snake venom.
