Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.
C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.
Kozono
H
H
Department of Bacteriology, Osaka University Medical School, Japan.
Kinoshita
T
T
Kim
Y U
YU
Takata-Kozono
Y
Y
Tsunasawa
S
S
Sakiyama
F
F
Takeda
J
J
Hong
K
K
Inoue
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003167:Complement Activation; D050577:Complement C3-C5 Convertases / Q000378:metabolism*; D003179:Complement C3b / Q000302:isolation & purification / Q000378:metabolism*; D015935:Complement C4b / Q000302:isolation & purification / Q000378:metabolism*; D003171:Complement Pathway, Classical; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding
D003179:Complement C3b; D015935:Complement C4b; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases