Synthesis of the branched form of erythroglycan by Friend GM 979 erythroleukemic cells

T. Kaizu, University of Kentucky
S. J. Turco, University of Kentucky
J. S. Rush, University of Kentucky
R. A. Laine, University of Kentucky

Abstract

Mouse GM979 erythroleukemic cells were found to synthesize the branched form of erythroglycan, a large polylactosamine structure known to be attached to band 3 and possibly to other proteins on human erythrocytes. Total protein-derived oligosaccharides from GM979 cells, labeled metabolically with [3H]glucosamine, [3H]mannose, or [3H]galactose, were prepared by hydrazinolysis after extracting the lipids. The 3H-labeled oligosaccharides were fractionated on Sephadex G-50 revealing 10-25% of each labeled product as a high molecular weight fraction (M(r)=10,000). Digestion of this [3H]glucosamine fraction with endo-β-galactosidase from Escherichia freundii, specific for the repeating structure of Galβ(1→4)GlcNAcβ(1→3), resulted in the following four products upon Bio-Gel P-2 Gel filtration; 1) a disaccharide with the structure GlcNAcβ(1→3)Gal,2) a trisaccharide with the structure Galβ(1→4)GlcNAcβ(1→3)βGal,3) a tetrasaccharide with the sequence Fucα(1 → 2)Galβ(1 → 4)GlcNacβ(1→3)Gal, and 4) a large complex fragment which contained mannose, glucosamine, galactose, and fucose (presumably the protein linkage region). Methylation linkage analysis of the large complex fraction shows mainly the presence of 4-substituted and terminal N-acetylglucosamine; 3,6-substituted, 6-substituted, 2-substituted, and 2,3-substituted galactose. The GM979 cell erythroglycan is only 30% susceptible to endo-β-galactosidase degradation probably because of the branched galactose residues, whereas the linear form of erythroglycan from human K562 cells is 60% susceptible. The branched residues in GM979 cell saccharides indicate that this mouse cell line bears an arborized erythroglycan-like glycopeptide similar to those found on human adult erythrocytes, and thus may be a source for the enzyme which transfers an N-acetylglucosamine residue to a 3-linked galactose to form a 3,6-disubstituted galactose.