Use of a monoclonal antibody (GA3) to demonstrate lineage restricted O-glycosylation on leukosialin during terminal erythroid differentiation

Abstract

A murine monoclonal antibody (GA3) obtained by immunizing mice with cells of the human erythroleukemic cell line K562 is shown to define a 105 kilodalton (kd) membrane antigen on K562 cells that is restricted within the hematopoietic system to the erythroid lineage and to a minor population of CD3, CD4 positive T lymphocytes. Cocapping studies and immunoprecipitation experiments performed with GA3 and L10, an anti-sialophorin monoclonal antibody reacting with leukosialin (Gp 105) on K562 cells, demonstrate that the antigen detected by GA3 on K562 cells is identical to leukosialin. Neuraminidase treatment but not tunicamycin treatment of K562 cells abolishes the expression of the GA3-epitope without affecting the L10-epitope thus providing evidence that terminal sialic acid present on O-linked oligosaccharide chains on Gp 105 is essential for the expression of the GA3-epitope. Further analysis by flow cytometry and immune panning experiments performed on bone marrow cells with GA3 or L10 demonstrate that, in contrast to L10, which reacts with all types of hematopoietic progenitors, the epitope recognized by GA3 is restricted to the erythroid lineage, and appears during erythroid differentiation before glycophorin A on the earliest morphologically recognizable erythroid precursor, the proerythroblast. Our results therefore suggest that O-linked oligosaccharides on leukosialin express lineage restricted and even maturation restricted antigenic structures that might serve as cell lineage specific markers.