THE C1Q-BINDING CELL-MEMBRANE PROTEINS CC1Q-R AND GC1Q-R ARE RELEASEDFROM ACTIVATED CELLS - SUBCELLULAR-DISTRIBUTION AND IMMUNOCHEMICAL CHARACTERIZATION

Two types of widely coexpressed cell surface C1q-binding proteins (C1q -R): a 60-kDa calreticulin-homolog which binds to the collagen-like '' stalk'' of C1q and a 33-kDa protein with affinity for the globular ''h eads'' of the molecule, have been described. In this report, we show t hat the two molecules are also secreted by Raji cells and peripheral b lood lymphocytes and can be isolated in soluble form from serum-free c ulture supernatant by HPLC purification using a Mono-Q column. The two purified soluble proteins had immunochemical and physical characteris tics similar to their membrane counterparts in that both bound to inta ct C1q and to their respective C1q ligands, cC1q and gC1q. In addition , N-terminal amino acid sequence analyses of the soluble cC1q-R and gC 1q-R were found to be identical to the reported sequences of the respe ctive membrane-isolated proteins. Ligand blot analyses using biotinyla ted membrane or soluble cC1q-R and gC1q-R showed that both bind to the denatured and nondenatured A-chain and moderately to the C-chain of C 1q. Moreover, like their membrane counterparts, the soluble proteins w ere found to inhibit serum C1q hemolytic activity. Although cC1q-R was released when both peripheral blood lymphocytes and Raji cells were i ncubated in phosphate-buffered saline for 1 hr under tissue culture co nditions, gC1q-R was releasable only from Raji cells, suggesting that perhaps activation or transformation leading to immortalization is req uired for gC1q-R release, Subcellular fractionation of Raji cells and analyses by enzyme-linked immunosorbent assay and Western blotting sho wed that the two molecules are present in the cytosolic fractions as w ell as on the membrane. The data suggest that soluble forms of both C1 q-binding molecules are released from cells and that these molecules m ay play important roles in vivo as regulators of complement activation . (C) 1997 Academic Press.