Construction and expression of a novel recombinant anaphylatoxin, C5a-N19, as a probe for the human C5a receptor.
We have constructed a novel recombinant C5a anaphylatoxin (C5a-N19) containing a 19-residue amino-terminal extension peptide, using a plasmid vector which secretes the nascent polypeptide to the Escherichia coli periplasmic space. C5a-N19 was purified from cell lysates by immunoaffinity chromatography using a monoclonal antibody which recognizes a portion of the amino-terminal extension peptide. C5a-N19 was characterized as biologically indistinguishable from the unmodified recombinant anaphylatoxin for release of lysosomal enzymes from dibutyryl-cAMP-differentiated U937 cells. In contrast to unmodified C5a, which is not recognized by anti-C5a antibodies following binding to its cellular receptor, receptor-bound C5a-N19 is recognized by the monoclonal antibody directed against the amino-terminal extension sequence. Because the monoclonal antibody recognizes the C5a-receptor complex on cells, this methodology is useful in fluorescence sorting of C5a receptor-positive cells. A C5a receptor affinity column was constructed by saturating monoclonal antibody bound to agarose with C5a-N19. Digitonin-solubilized C5a receptor from dibutyryl-cAMP-induced U937 cells was adsorbed to the matrix and eluted by dissociation of the ligand-receptor complex from the antibody. Analysis by SDS-polyacrylamide gel electrophoresis revealed a unique protein band at 41K, consistent with the molecular weight predicted from cross-linking experiments when the contribution of C5a is subtracted. Development of this recombinant C5a derivative provides a useful probe previously unavailable for the C5a receptor molecule.
Gerard
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Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
Gerard
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D000706:Anaphylatoxins; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D008969:Molecular Sequence Data; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins
D000706:Anaphylatoxins; D000911:Antibodies, Monoclonal; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins