Sialyl Le x structures in O-glycans attached to lysosomal membrane glycoproteins, lamp-1 and lamp-2. Comparison to those in N-glycans.

Abstract

Poly- N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids (Fukuda, M. (1994) In Molecular Glycobiology, (ed. M. Fukuda and O. Hindsgaul), pp. 1–52, Oxford University Press, Oxford). However, the extent of poly- N-acetyllactosamine formation between O-glycans and N-glycans in a given glycoprotein has not been determined. Attempts were made in HL-60 cells to determine the amount of sialyl Le x structures in O-glycans and N-glycans attached to the major lysosomal membrane glycoproteins, lamp-1 and lamp-2. Lamp molecules were immunoprecipitated from 3H-glucosamine labeled HL-60 cells. Glycopeptides were prepared by pronase or trypsin digestion, and O-glycan containing glycopeptides were isolated by affinity chromatography using Jacalin-Agarose. The glycopeptides bound to Jacalin-Agarose were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Similarly, the glycopeptides unbound to Jacalin-Agarose, which represent N-glycans, were isolated by Sephadex G-50 gel filtration. Sequential glycosidase digestion of the O- and N-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following features: (1) each lamp-1 and lamp-2 molecule contains about 2 and 1 poly- N-acetyllactosaminyl O-glycan chains, (2) about 65% of these poly- N-acetyllactosaminyl O-glycans contain sialyl Le x termini, NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAcβ1→R, and (3) N-glycans and O-glycans are modified in almost equal efficiency to express sialyl Le x structures. These results indicate that in lamp molecules both O-glycans and N-glycans contribute to the expression of sialyl Le x structures.