Biochemical characterization of the 44G4 antigen from the HOON pre-B leukemic cell line.
The 44G4 antigen is expressed in high amounts on human endothelial cells and at low levels on leukemic cells of pre-B and myelomonocytic origin. Its level of expression on the pre-B leukemic HOON cell line used for derivation of the corresponding mAb is intermediate but sufficient to permit the purification of the Ag. The molecule isolated by immunoaffinity from HOON is a glycoprotein since it bound to Ricinus communis agglutinin, wheat germ agglutinin, and peanut agglutinin lectins. The Ag was purified 2400-fold from a soluble taurocholate extract of HOON cells by affinity to wheat germ agglutinin-agarose and 44G4-IgG-Sepharose. The purified glycoprotein is likely a homodimer as it migrated on SDS-PAGE with an apparent m.w. of 170,000, nonreduced, and 95,000, reduced. Removal of N-linked oligosaccharides by endoglycosidase F led to a decrease in m.w. of 25,000; if neuraminidase and O-glycanase were also present, the total decrease in m.w. was 33,000 suggesting a polypeptide chain of 62,000 and 8,000 in O-linked substitutions. The glycoprotein digested with N-glycanase, neuraminidase, or O-glycanase could still be immunoprecipitated with the 44G4 mAb indicating that the antigenic epitope resides in the polypeptide. By Western blot analysis, the dissociated but nonreduced protein was reactive with 44G4, whereas the reduced and alkylated protein was not. Therefore, the epitope is dependent on the presence of intact disulfide bond(s). Sequential immunoprecipitation with OKT9 and 44G4 antibodies indicated that these epitopes are present on two distinct molecules and that 44G4 is distinct from the transferrin receptor despite a similar subunit structure.
Gougos
A
A
Division of Immunology, Hospital for Sick Children, Toronto, Ontario, Canada.
Letarte
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D054278:Embryonal Carcinoma Stem Cells; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000276:immunology; D007945:Leukemia, Lymphoid / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D014411:Neoplastic Stem Cells / Q000276:immunology; D010455:Peptides / Q000276:immunology; D011487:Protein Conformation; D011990:Receptors, Transferrin / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D037102:Lectins; D010455:Peptides; D011990:Receptors, Transferrin