Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins.
HL-60 cells were induced to differentiate into granulocytic cells by dimethyl sulfoxide, and structures of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins (lamp-1 and lamp-2) were elucidated before and after differentiation. Lamp-1 and lamp-2 were immunoprecipitated from the cells after labeling with radioactive sugars, and glycopeptides were prepared. The structures of glycopeptides obtained after serial lectin-affinity chromatography were elucidated by endo-beta-galactoside and methylation analysis. Glycopeptides bound to tomato lectin-Sepharose were found to be tetraantennary oligosaccharides that contain two or three poly-N-acetyllactosaminyl chains, of which one side chain contains three or more N-acetyllactosaminyl repeats, whereas those bound to Datura stramonium agglutinin-Sepharose were found to be tetraantennary oligosaccharides containing one or two short poly-N-acetyllactosaminyl side chains. Glycopeptides that were not bound to concanavalin A, tomato lectin, or D. stramonium agglutinin were found to be triantennary oligosaccharides with a negligible amount of poly-N-acetyllactosaminyl side chains. Comparison of Asn-linked oligosaccharides from undifferentiated and differentiated HL-60 cells reveals the following features. First, the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl side chains increases dramatically with a concomitant decrease in less complex Asn-linked oligosaccharides after differentiation. Second, the number of poly-N-acetyllactosaminyl side chains per Asn-linked oligosaccharides increases significantly. These increases in poly-N-acetyllactosamine were associated with increased activity of UDP-GlcNAc:beta-D-Gal-beta 1----3-N-acetylglucosaminyltransferase "extension enzyme," a key enzyme in the formation of poly-N-acetyllactosamines. Furthermore, the increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 resulted in longer half-lives of lamp-1 and lamp-2 in differentiated HL-60 cells. These results suggest strongly that the differentiation of HL-60 cells into more phagocytic cells is associated with an increase in the complexity of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins, which in turn may play a role in stabilizing lysosomes.
Lee
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La Jolla Cancer Research Foundation, Cancer Research Center, California 92037.
Wang
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WC
Fukuda
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D015703:Antigens, CD; D001216:Asparagine; D002240:Carbohydrate Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002846:Chromatography, Affinity; D005944:Glucosamine / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D011134:Polysaccharides / Q000378:metabolism*
D015703:Antigens, CD; D006020:Glycopeptides; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D001216:Asparagine; C037199:poly-N-acetyllactosamine; D008715:Methionine; D006602:Hexosyltransferases; D005944:Glucosamine