Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines.

S R Carlsson,H Sasaki,M Fukuda

doi:10.1016/s0021-9258(18)67162-5

Abstract

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.

Highlights

Structures of 0-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoidcell lines wereexamined
Leukosialin was isolated by specific immunoprecipitation from cellswhichwere metabolically labeled with [3H]glucosamine,and glycopeptides were isolated after Pronase digestion. 0-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestionS, mith degradation, and methylation anaylsis
These glycoproteins isolated from different cell lines show significant differences in molecular weight,as estimatedby SDS-polyacrylamide gel electrophoresis, their polypeptide portions appear tobe very similar or the same, based on their size and reactivity with antibodiesT. hese glycoproteins, termed leukosialin, were shown to containa large number of 0-linked oligosaccharides and one asparagine-linkedoligosaccharide/molecule [1]

Summary

To whom correspondence should be addressed La Jolla Cancer

Preparation of 0-Linked Oligosaccharidesfrom Leukosialin Glycopeptides-The Pronase-digested material was applied to a column (1.0 X 110 cm) of Sephadex G-50 (superfine) equilibrated with 0.1 M NH4HC03. The pH of the samples was neutralized by adding methanol containing acetic acid and lyophilized after addition of water. The column was washed with 0.2 M sodium phosphate buffer, pH 7.4, in order to elute highly charged materials. Treatment with Escherichia coli @-galactosidase (20 units) was done in 80 p1 of 50 mM sodium phosphate buffer, p H 7.3, containing 4 mM MgC12. The periodate-oxidized fractions containing oligosaccharides, detected by radioactivity, were pooled and lyophilized.The periodate-oxidizedborohydride-reducedsampleswerethenhydrolyzed in 0.02 N HCI a t 80 "Cfor 1 h. Peak I and peak I1 from both K562 andHL-60 cells were examined by SDS-

RESULT?

DISCUSSION

Vibri oc holer an euraminidas et reatwent