Vipera lebetina factor X activating enzyme – cloning and sequencing

Abstract number: P1096

Siigur E., Aaspollu A., Trummal K., Tammiste I., Siigur J.

National Institute of Chemical Physics & Biophysics, Estonia

Vipera lebetina venom contains several enzymes interfering with coagulation process. V. lebetina factor X activator (VLFVA) has been isolated and characterized [Siigur et al. BBA 2001; 1568: 90–8]. It is a three-subunit enzyme consisting of heavy chain (HC, 57.5 kDa) and two light chains (LC1, 17.4 kDa, and LC2, 14.5 kDa) connected with disulfide bridges. Its amino acid sequences are deduced from the cDNAs encoding the enzyme, for elucidation of the biosynthesis and post-translational processing of the protein. The cDNAs were obtained by screening the V. lebetina venom gland cDNA library by plaque hybridization and PCR techniques. Two individual sequences encoding the heavy chain and light chain LC1 were established. The cDNA corresponding to heavy chain (2346 nucleotides) encodes an open reading frame of 612 amino acids including signal peptide, propeptide and mature enzyme regions, the last one (425 amino acids) comprising metalloprotease, disintegrin-like and cysteine-rich domains and three putative Asn-glycosylation sites. The light chain LC1 (714 nucleotides) is processed from a particular gene comprising 5'-UTR, signal peptide and mature protein coding regions, and 3'-UTR. In translated sequence the 23-amino acid signal peptide is followed by 123-amino acid lectin-like protein (one putative Asn-glycosylation site) that is processed post-translationally. Based on N-terminal protein sequencing, the second light chain LC2 has heterogeneous N-terminal that is not included in the sequence of LC1. Thus, LC2 represents an individual protein, not an LC1 isoform or truncated form. The accordance of the translated sequence to the native enzyme sequence was proved by comparison of the masses of tryptic fragments obtained from the native enzyme with the calculated masses from translated sequences by MALDI TOF mass spectrometry for both heavy chain and LC1. Vipera lebetina factor X activator is the first so-called P-IV class protein cloned and the main result suggests that all subunits are synthesized from individual genes.