Primary structure and autoproteolysis of brevilysin H6 from the venom of Gloydius halys brevicaudus.
The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.
Fujimura
S
S
Department of Chemistry, Faculty of Science, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
Oshikawa
K
K
Terada
S
S
Kimoto
E
E
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002384:Catalysis; D003435:Crotalid Venoms / Q000737:chemistry*; D019483:Disintegrins / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry*; D045726:Metalloproteases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000737:chemistry; D016415:Sequence Alignment; D014688:Venoms / Q000737:chemistry*; D014757:Viper Venoms / Q000737:chemistry
D002135:Calcium-Binding Proteins; D003435:Crotalid Venoms; D019483:Disintegrins; D004220:Disulfides; D011498:Protein Precursors; D014688:Venoms; D014757:Viper Venoms; D045726:Metalloproteases; D008666:Metalloendopeptidases; C413765:brevilysin H6 protein, Gloydius halys brevicaudus; C118463:brevilysin L6