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.
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 NH2-(XN18) and COOH-(XC15) terminus of gC1q-R, and (3) bind to microtiter well fixed intact Raji cells. Several clones were identified: MAbs 46.23 and 60.11 (IgG1 kappa), 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 (IgG1 kappa) 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.
Department of Medicine, State University of New York, Stony Brook 11794, USA.
B
B
Ghebrehiwet
P D
PD
Lu
W
W
Zhang
B L
BL
Lim
P
P
Eggleton
L E
LE
Leigh
K B
KB
Reid
E I
EI
Peerschke
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002352:Carrier Proteins; D015922:Complement C1q / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011951:Receptors, Complement / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C087299:C1QBP protein, human; C490193:C1qbp protein, mouse; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D011951:Receptors, Complement; C032402:complement 1q receptor; D015922:Complement C1q