Synthesis of the branched form of erythroglycan by Friend GM979 erythroleukemic cells.

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 (Mr = 10,000). Digestion of this [3H]glucosamine fraction with endo-beta-galactosidase from Escherichia freundii, specific for the repeating structure of Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3), resulted in the following four products upon Bio-Gel P-2 Gel filtration: 1) a disaccharide with the structure GlcNAc beta (1 leads to 3)Gal, 2) a trisaccharide with the structure Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3) beta Gal, 3) a tetrasaccharide with the sequence Fuc alpha (1 leads to 2)Gal beta (1 leads to 4)GlcNAc beta (1 leads to 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-beta-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.