Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies

Abstract

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q--immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15--0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9--12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338--344].

Chemical verification of the C1q receptor site on IgG

“…The modification was minimal to ensure that there was: (1) no alteration in either conformational integrity or the antigen binding capacity of the IgG molecules measured by CD spectra and precipitin curves; (2) specific labelling of few lysine residues. The specific activity of the modifying acetate groups indicated that there was 1.2 + 0. agree with our results for acetic anhydride modified IgC where 2.2 + 0.3 modified lysine residues on IgG changed the binding affinity 29-fold [21]. This is a significant change in binding affinity since modifications of arginine and tryptophan residues have no effect on Clq binding affinity for IgG.…”

“…Immune rabbit IgG (anti-ovalbumin) was prepared asin [21] thenmodified with [ 1-14C]aceticanhydride which reacts with lysine residues [ 19,201. The conditions of modification, acetate buffer at pH 5 .O and O"C, are extremely mild and do not lead to protein denaturation.…”

“…Comparison of precipitin curves and circular dichroism spectra for the modified and control IgG were also as in [21].…”

“…The binding of 12SI-labelled Clq to these aggregates was measured by a modification [21] of the method in [33].…”

Conformational changes in C1q upon binding to IgG oligomers

“…The binding of lZSl-labelled Clq to immune complexes was measured using the methods of [7]. The fluorescence of protein-bound ANS was measured at 490 _+ 5 nm, following excitation at 390 _+ 5 nm, using an Aminco SPF-500 spectrofluorimeter, with the cell-holders thermostatted at 37°C.…”