Identification of functional domains on gC1Q-R, a cell surface protein that binds to the globular 'heads' of C1Q, using monoclonal antibodies and synthetic peptides

Abstract

A membrane protein (33 kDa) that binds to the globular 'heads' of C1q (gC1q-R) has been recently described. The full length cDNA encoding gC1q-R has been cloned, expressed in E. coli and using the purified recombinant protein (rgC1q-R) as an immunogen, a panel of IgG monoclonal antibodies (MAb) has been produced by fusion of spleen cells from hyperimmunized BALB/c mice with NSO mouse myeloma partners. From this fusion, 60 anti-gC1q-R hybridomas were selected and evaluated for their ability to (1) discriminate between the mature form (MF) of gC1q-R (residues 74-282) and a truncated form (TF) lacking residues 74-95, which contains a major C1q binding site, (2) recognize two functionally defined synthetic peptides derived from the NH 2- (XN 18) and COOH-(XC 15) terminus of gC1q-R, and (3) bind to microtiter well fixed intact Raji cells. Several clones were identified: MAbs 46.23 and 60.11 (IgG(1κ)), reacted strongly with ELISA plate-fixed intact Raji and K562 cells, MF, and the XN 18 peptide, but had poor or no reactivity with TF; MAbs 74.5.2 > 25.15 (IgG(1κ)) recognized both MF and TF and are directed against epitopes in the XC 15 peptide that contains a binding site for high- molecular-weight kininogen and Factor XII.