Abstract
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.
Highlights
Susumu Tsunasawa(I, From the $Department of Bacteriology, Osaka University Osaka Medical College, Takatsuki, Osaka and II Division Suitu, Osaka, Japan
C3b Complex-For establishment of a method to obtain a sufficient amount of the C4b.C3b complex for analysis of its primary structure, the classical pathway was activated on antibody-sensitized sheep erythrocytes (EA) in human serum
We first examined whether the covalent C3b-binding site was restricted to a small region of C4b or was randomly distributed
Summary
Susumu Tsunasawa(I , From the $Department of Bacteriology, Osaka University Osaka Medical College, Takatsuki, Osaka and II Division Suitu, Osaka, Japan. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. An M, 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. 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. C5 convertases are assembled on the surface of complement targets, such as microorganisms, during activation of the pathways and play roles in elimination of the targets by generating C5a and C5b from C5 [1] and perhaps by acting as ligands of the complement receptor CR1 present on phagocytes and other cells [5, 6]
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