Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom

Abstract

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142–152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30 min at pH 8.5 and 37 °C in the absence of Ca 2+. Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser 60–Asn 61, Thr 99–Ala 100, and Phe 103–Asp 104 bonds may trigger the autoproteolysis. Addition of Ca 2+ completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.