Abstract
The immunoglobulin G binding site in the globular regions of human complement subcomponent C1q has been investigated by chemical modification of histidine residues with diethylpyrocarbonate and arginine residues with phenylglyoxal and cyclohexane-1,2-dione (CHD). Only the modification of arginine residues with CHD fulfills the requirements of a specific modification without unwanted side reactions. Specific modification of arginine residues with CHD results in loss of immune complex recognition without affecting the binding of C1r2S2 to form C1. The gross structure of C1q is not changed by CHD treatment, and immune complex binding is restored to 82% of the control upon NH2OH treatment. Enzymic digestion and isolation of the modified peptides indicate that the modification by CHD of 4 to 5 arginine residues (A162, B114, B129, C156, and possibly B163) per C1q globular "head" abolishes the ability of C1q to interact with immune complexes. These residues define two areas (and possible binding sites for IgG) on the globular region of C1q: B114-B129 (site 1) and A162-(B163)-C156 (site 2). Sequence comparison and solvent exposure predictive studies favor site 2 as the immunoglobulin G binding site on the globular regions of C1q, although the participation of site 1 cannot be ruled out.
Highlights
Arginine Residues of the Globular Regions of Human C l q Involved in the Interaction with Immunoglobulin G*
Enzymic digestion and isolation of the modified peptides indicate that the modification by CHD of 4 to 5 arginine residues (A162, B114, B129, C156a,nd possibly B163) per Clq globular “head”abolishes the ability of Clq to interact with immune complexes
The Clq binding site inFtche region of IgG has been mapped to the COOH-terminal half of the Cy2 domainby genetic engineering [10].Substitution by site-directedmutagenesis of residues Glu318,LYS~~O, aLnyds322by Ala abrogates C l q binding by a mouse monoclonal IgG2b [11], supporting therole of the two COOH-terminal @-strandsof the Cy2 domain as part of the residues define two areas
Summary
Clq Hemolytic Assay-The Clq hemolytic assay was performed as in Tenner et al [21]. Clq-depleted serum (RQ) contained less than 0.05% original Clq hemolytic activity, and total complement titer (CH50) was restored to 90% of the orig-inal serum upon addition of purified Clq. DHCH-Arg-containing peptides were detected employingthe GirardT reagent as described by Patthy et al [36]. After 4h at 37 "C with occasional shaking, the digestion was stopped with 200 p1 of acetic acid and thedigestion mix was loaded in a Bio-Gel P-6 (200-400 mesh, Bio-Rad) gel filtration column (115 X 1.5 cm) equilibrated in 20 mM acetic acid containing 100 mM NaCl. Sample was eluted at 11 ml/h, and 1-ml fractions collected.A280nm, and DHCH-Arg (expressed as the difference in AsW,, in the Girard-Tassay) were measured in the chromatographic fractions. HPLC Purification of DHCH-Arg-containing Peptides-A Waters (Millipore) chromatograph was employed for reversed-phase separation of peptides using different columns, solvent combinations, and gradients? Human Clq sequence data used for determining the position of the analyzed peptides were taken from Sellar et al [5], and each chain was independently numbered. No modification of the gross antigenic structure of C l q was detected by doubleimmunodiffusion (not shown)
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