Structure and function of human C5a anaphylatoxin. Selective modification of tyrosine 23 alters biological activity but not antigenicity.
Reaction of either human C5a or its des-Arg74 derivative (des-Arg74-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyrosyl23-C5a and -des-Arg74-C5a were separated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyrosyl23-C5a and -des-Arg74-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a derivatives serving as competing ligands and a murine antihuman C5a monoclonal antibody employed as first antibody demonstrated that selective modification of tyrosine23 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyrosyl23-C5a was approximately 3-fold less active than native C5a in both bioassays and competitive ligand-receptor binding assays. Additionally, photoreactive derivatives prepared by coupling either N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate to aminotyrosyl23-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These observations suggest that the tyrosyl23 residue of C5a may participate importantly in the binding interactions of this chemotactic factor and its granulocyte receptor.
R J
RJ
Johnson
D E
DE
Chenoweth
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D003176:Complement C3 / Q000302:isolation & purification*; D015926:Complement C3a; D003182:Complement C5 / Q000378:metabolism*; D015936:Complement C5a; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006801:Humans; D009504:Neutrophils / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis; D010782:Photolysis; D011863:Radioimmunoassay; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D003176:Complement C3; D003182:Complement C5; D000939:Epitopes; D010446:Peptide Fragments; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D014443:Tyrosine; D015926:Complement C3a; D015936:Complement C5a; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D013774:Tetranitromethane