Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase.
We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the alpha chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade.
Yamamoto
Chieko
C
Faculty of Engineering, Sojo University, Kumamoto, Japan. yamamoto@nephro.ne.jp
Tsuru
Daisuke
D
Oda-Ueda
Naoko
N
Ohno
Motonori
M
Hattori
Shosaku
S
Kim
Sung-Teh
ST
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003167:Complement Activation / Q000187:drug effects; D003176:Complement C3 / Q000737:chemistry / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000276:immunology*; D015926:Complement C3a / Q000378:metabolism; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D003435:Crotalid Venoms / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D012697:Serine Endopeptidases / Q000276:immunology* / Q000302:isolation & purification; D012911:Snakes; D013917:Thrombin / Q000276:immunology; D014357:Trypsin / Q000276:immunology
D003176:Complement C3; D015938:Complement Membrane Attack Complex; D003435:Crotalid Venoms; D015926:Complement C3a; D050577:Complement C3-C5 Convertases; D012697:Serine Endopeptidases; C057255:flavoxobin; D014357:Trypsin; D013917:Thrombin