Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain.
Molecular cloning has revealed the DNA sequence of the human receptor for the C5a anaphylatoxin (C5aR). In this study, mAb and polyclonal antibodies with specificities for deduced hydrophilic sequences of the receptor protein were employed to determine the expression and the topography of C5aR on PBL. Evidence was obtained that an antigenically homogenous receptor exists on human neutrophils, eosinophils, and monocytes that binds C5a and C5a(desArg). The assignment of epitopes to extra- or intracellular receptor domains as determined by FACS analysis of intact or permeabilized cells confirmed the general topography of the C5aR that had been predicted by hydropathy analysis. Competitive binding studies were performed to examine whether extracellular receptor domains participate in ligand binding. The occupation of the receptor by C5a inhibited only the binding of antibodies that were specific for the receptor's aminoterminal domain (C5aR-EX1). The anti-EX1 mAb S5/1 effectively antagonized C5a/C5a(desArg) biological activity. A heptameric peptide (D15DKDTLD21) was identified as the smallest receptor fragment that was recognized by the mAb S5/1 or by polyclonal rabbit anti-EX1 lg. These results imply that an amino acid sequence rich in aspartate within the receptor aminoterminus represents both an immunodominant epitope and a ligand binding site on the C5aR.
Oppermann
M
M
Department of Immunology, University of Göttingen, FRG.
Raedt
U
U
Hebell
T
T
Schmidt
B
B
Zimmermann
B
B
Götze
O
O
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D015936:Complement C5a / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a