Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom.
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 30min at pH 8.5 and 37 degrees 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.
Deshimaru
Masanobu
M
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. deshi@fukuoka-u.ac.jp
Ichihara
Makoto
M
Hattori
Takahiro
T
Koba
Kumiko
K
Terada
Shigeyuki
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019295:Computational Biology; D003435:Crotalid Venoms / Q000201:enzymology / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D019483:Disintegrins / Q000235:genetics* / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017819:Viperidae; D015032:Zinc / Q000378:metabolism
D003435:Crotalid Venoms; D017931:DNA Primers; D018076:DNA, Complementary; D019483:Disintegrins; D004792:Enzyme Precursors; D045726:Metalloproteases; D015032:Zinc