Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.
The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.
Department of Biochemistry, University of Oxford, United Kingdom.
A
A
Sepp
A W
AW
Dodds
M J
MJ
Anderson
R D
RD
Campbell
A C
AC
Willis
S K
SK
Law
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003167:Complement Activation; D015934:Complement C4a / Q000378:metabolism; D015935:Complement C4b / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004952:Esters / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection
D004952:Esters; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D015934:Complement C4a; D015935:Complement C4b; D005990:Glycerol; D005998:Glycine