The immunoglobulin G binding site of complement protein C1q.

The involvement of Clq lysine residues in the binding of human Clq to rabbit IgG was assessed by chemical modification. Modification on of Clq lysyls with acetic anhydride was complicated by the lack of selectivity of the reagent, as loss of Clq functional activity, due to the modification of tyrosine residues, occurred bcfore any effect due to lysyl modification was apparent. Treatment of Clq with trinitrobenzene sulfonate or fluorescein isothiocyanate resulted in loss of binding activity but was also associated with a Clq concentration dependent precipitation of Clq suggesting structural alteration of the protein. N-(iodoacetylaminoethyl) -8 naphthylamine-l-sulphonate was found to be a non-covalent inhibitor of the interaction between Clq and antigen-aggregated IgG. Crosslink ing of human Clq to antigen-aggregated IgG by treatment with dimethyl -3,3'-di thiobispropionimidate (Lysyl-Lysyl crosslinking), 1,5difluoro- 2,4—dinitrobenzene (Lysyl—, Tyrosyl-, Histidyl-) or l-ethyl-S- (Bdimethyl aminopropyl) carbodiimide (Lysyl-Carboxyl) was found to involve the disulfide-linked AB and CC chains of Clq. As the DTBP crosslinking involved linking lysyl groups, the distribution of the lysine residues restricted the possible sites for crosslinking. Possible candidate lysyls in the globular head region of the C chain are limited to four residues flanking the interchain disulfide bond, which are thus likely to be close in space and may constitute a single contiguous contact site for IgG. This region is of high identity between the C and B chains. 1—ethyl-3 -(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking was also determined to involve lysyl groups of Clq. This strengthened the assignment of the proposed lysine residues as points of contact with IgG as the chemistry of carbodiimide crosslinking is a " zero-length" process introducing no additional atoms between the participating sidechain s. This crosslinking method also indicated that iodination of Clq may inactivate the Ig binding activity associated with the C chain into which the 125I-label is incorporated. All three crosslinking methods gave evidence for an altered conformation of Clq bound to antigen-aggregated IgG. Only when C1q bound to immune complexes was treated with crosslinkers was crosslinking within the disulfide-linked AB dimer apparent by the appearance, on Western blotting of non-reduced SDS-PAG E samples, of a more rapidly migrating band with A/ B chain reactivity. Direct identification of the crosslinking sites by proteolysis of the covalent complex formed by DTBP-treatment of Clq and aggregated IgG and purification of the fragment containing crosslinks was unsuccessful. Two anti-Clq monoclonal antibodies, BUS—1 and BUS-2, which inhibited the binding of Clq to antigen aggregated IgG were produced. These mAbs competitively inhibited the binding of each other to Clq and so probably recognised the same or overlapping epitopes. Western blotting analysis showed BUS-1 to have weak reactivity with both the disulfide-linked AB and CC chains, but not with the reduced and alkylated free A, B or C chains. Reduction of the accessible disulfides of 125I—labeled C1q, under non-denaturing conditions, diminished the binding of Clq to immobilized BUS-1. These data were consistent with the BUS—1 epitope being discontinuous and occurring in the homologous regions of both the C and B chains flanking the intrachain disulfides. This interpretation was supportive of the conclusions of the crosslinking study from which it was also proposed these regions of Clq contact IgG. The modification of tyrosine residues of Clq decreased both the IgG-binding activity of the protein and its reactivity with the BUS-1 mAb. This was consistent with the contention that the mAb binds to the Ig recognition site. The loss of both BUS-1 reactivity and IgG binding activity on the cleavage of the accessible cysteines of Clq also indicates some degree of identity between the Ig recognition site and the BUS—1 epitope. A functionally active Fab/c fragment of IgG (i.e. consisting of one antigen-binding Fab and one complement interactive Fc) was produced by the use of cyanocysteine chemistry to achieve cleavage of the heavy chain polypeptide in the hinge region. This fragment is of value as a potential therapeutic agent for the activation of effector functions in situ as it is not subject to antigenic modulation. The physicochemical basis of the enhancement of Clq binding to immune complexes in the presence of polyethylene glycol was shown to be an excluded volume effect. This is of interest as Clq binding activity in the presence polyethylene glycol forms the basis of some assays for circulating immune complexes