Identification of Functional Domains on gClQ-R, a Cell Surface Protein That Binds to the Globular "Heads" of C1Q, Using Monoclonal Antibodies and Synthetic Peptides

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Abstract
A membrane protein (33 kDa) that binds to the globular "heads" of Clq (gClq-R) has been recently described. The full length cDNA encoding gClq-R has been cloned, expressed in E. coli and using the purified recombinant protein (rgClq-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-gClq-R hybridomas were selected and evaluated for their ability to (1) discriminate between the mature form (MF) of gClq-R (residues 74-282) and a truncated form (TF) lacking residues 74-95, which contains a major Clq binding site, (2) recognize two functionally defined synthetic peptides derived from the NH2-(XN18) and COOH-(XC15) terminus of gClq-R, and (3) bind to microtiter well fixed intact Raji cells. Several clones were identified: MAbs 46.23 and 60.11 (IgG1κ, reacted strongly with ELISA plate-fixed intact Raji and K562 cells, MF, and the XN18 peptide, but had poor or no reactivity with TF; MAbs 74.5.2 > 25.15 (IgG1κ) recognized both MF and TF and are directed against epitopes in the XC15 peptide that contains a binding site for high-molecular-weight kininogen and Factor XII.