Binding of C3b proceeds by a transesterification reaction at the thiolester site

Abstract

The binding of C3b, the opsonic fragment of the third component of complement (C3), to bacterial surface structures mediates two events important in host defence: assembly of the C5b-C9 lytic complex and opsonic recognition by phagocytic cells (for reviews see refs 1, 2). These interactions proceed through a labile binding site³ located on the α′ chain of the C3b molecule, with the resultant formation of a covalent oxy-ester bond^4,5 in which the acyl group is contributed by the protein. By exposure of a titratable sulphydryl group^6,7 an internal thiolester of native C3 has also been localized to the α′ chain. Studies with ¹⁴C-methylamine, a nucleophile which is inherently reactive with a thiolester, have further indicated a stoichiometric (1:1) and covalent interaction, again within the α′ chain^8–10. After reaction of the native protein with ¹⁴C-methylamine and radioalkylation of the exposed sulphydryl with ³H-iodoacetic acid, a 35-residue tryptic peptide has been isolated that yields the sequence -Cys⁹-Gly-Glu-Glu¹²- on Edman degradation; tritium counts are released at step 9 (S[³H]-(carboxymethyl)cysteine) and ¹⁴C counts at step 12 (γ-glutamyl[¹⁴C]methylamide)⁸. We now present data which directly demonstrate that the second glutamyl residue of the reactive thiolester can, on proteolytic cleavage of the protein, donate its carbonyl group in a transesterification reaction with appropriate acceptor molecules. These results provide a model for analysis of the interactions at the molecular level between surface constituents of microorganisms and C3b.