Combined use of lectin affinity chromatography and endo-beta-galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces.
The trypsin-sensitive glycopeptides from cell surfaces of a multipotential murine haemopoietic cell line (DE) have been studied using serial lectin affinity chromatography on columns of immobilized lentil lectin (LCA), concanavalin A (Con A), and wheat-germ agglutinin (WGA). WGA-binding material consisted of glycopeptides that failed to bind to LCA and Con A. Step elution from the WGA-column with 0.01-, 0.1-, 0.5- and 1.0 M N-acetyl-D-glucosamine yielded four affinity classes of glycopeptide (WGA-W, WGA-I, WGA-S and WGA-SS respectively). WGA-W, WGA-I and WGA-S contained both alkali-stable (N-linked) and alkali-labile (O-linked) carbohydrate on high molecular weight glycopeptides. The WGA-SS fraction contained only N-linked carbohydrate. N-linked glycopeptides isolated from each WGA-binding class differed in molecular size, relative N-acetylneuraminic acid content and affinity for Ricinus communis 120 agglutinin. endo-beta-Galactosidase digestion showed that these glycopeptides contained polylactosamine-type glycans. Gel filtration profiles of the enzyme treated materials were different for each WGA-binding population suggesting variation in branching patterns and/or substitution with fucose residues. Affinity chromatography has shown that the WGA binding molecules are the major glycopeptide group at DE cell surfaces.
Morris
A J
AJ
Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Withington, Manchester, U.K.
Gallagher
J T
JT
Dexter
T M
TM
D000606:Amino Sugars / Q000032:analysis*; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006020:Glycopeptides / Q000032:analysis; D006026:Glycoside Hydrolases; D006412:Hematopoietic Stem Cells / Q000032:analysis*; D037102:Lectins; D051379:Mice; D011975:Receptors, Mitogen / Q000378:metabolism; D001616:beta-Galactosidase
D000606:Amino Sugars; D006020:Glycopeptides; D037102:Lectins; D011975:Receptors, Mitogen; C027503:wheat germ agglutinin receptor; C032233:lactosamine; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase