Isolation and characterization of My23, a myeloid cell-derived antigen reactive with the monoclonal antibody AML-2-23.
In this study, we describe the isolation and characterization of My23, a human myeloid antigen defined by the monoclonal antibody (MoAb) AML-2-23. Cells of the HL-60 human promyelocytic cell line, when cultured in the presence of 1,25-dihydroxyvitamin D3 (calcitriol), express a surface protein of approximately 50 to 55 kilodaltons (Kd) which was immunoprecipitated with the AML-2-23 MoAb. Furthermore, after 2 days of exposure to calcitriol, HL-60 cells began to release My23 into culture medium, as determined by the ability of culture supernatant from these cells to block the binding of AML-2-23 to myeloid cells. My23 release was almost totally inhibited by incubation of cells at 4 degrees C, and was partially blocked by treatment of cells with cycloheximide or tunicamycin. The culture supernatant blocking factor, soluble My23, was identified as a 45 to 50 Kd protein by Western blot/immune overlay, using AML-2-23 and an 125I-labeled second antibody. My23, which was affinity-purified from culture supernatant, retained the ability to block AML-2-23 binding to myeloid cells. The affinity-purified antigen migrated on SDS-PAGE as a diffuse band in the m.w. range of 44 to 52 Kd. On treatment with endoglycosidase, the apparent m.w. of My23 decreased to approximately 40,000, indicating the presence of carbohydrate residues on My23. Serum from mice immunized with the purified antigen reacted with the same spectrum of myeloid cells as AML-2-23 MoAb, reacted with the My23 soluble protein in immunoblots, and competed with AML-2-23 for binding to myeloid cells. Binding of this antiserum to myeloid cells was blocked by cell supernatant from both monocytes and calcitriol-treated HL-60 cells, suggesting, along with results from m.w. determinations of the two preparations, that the soluble and cell surface forms of My23 are similar. Moreover, based on our finding that human plasma specifically inhibits the binding of AML-2-23 to myeloid cells, My23 may also be released in vivo. The enhanced expression of My23 on activated and more mature myeloid cells and its shedding or secretion by these cells is consistent with a functional role for My23.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D001798:Blood Proteins / Q000276:immunology; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003470:Culture Media; D006098:Granulocytes / Q000276:immunology*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000276:immunology*; D008970:Molecular Weight; D009000:Monocytes / Q000276:immunology*; D009439:Neuraminidase; D010447:Peptide Hydrolases
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001798:Blood Proteins; D003470:Culture Media; D009439:Neuraminidase; D010447:Peptide Hydrolases; D002117:Calcitriol