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Combined use of lectin affinity chromatography and endo‐β‐galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces
Author(s) -
Morris Andrew J.,
Gallagher John T.,
Dexter T. Michael
Publication year - 1986
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130010110
Subject(s) - wheat germ agglutinin , chemistry , lectin , glycopeptide , concanavalin a , affinity chromatography , fucose , biochemistry , agglutinin , glycan , microbiology and biotechnology , glycoprotein , chromatography , enzyme , biology , in vitro , antibiotics
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‐β‐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.