Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurensis lectin binding in a new Chinese hamster ovary cell line.
We report the characterization of two Chinese hamster ovary cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient cell mutant pgsA-745 with a cDNA library prepared from wild-type cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.
Bai
X
X
Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093-0687, USA.
Brown
J R
JR
Varki
A
A
Esko
J D
JD
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002460:Cell Line; D006224:Cricetinae; D005690:Galactose / Q000378:metabolism*; D046911:Macromolecular Substances; D010835:Phytohemagglutinins / Q000378:metabolism*; D037121:Plant Lectins; D011134:Polysaccharides / Q000378:metabolism*; D019685:Rosales / Q000201:enzymology / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010835:Phytohemagglutinins; D037121:Plant Lectins; D011134:Polysaccharides; D013431:Sulfates; C100156:leukoagglutinins, plants; D001216:Asparagine; D015238:Sulfotransferases; C097036:galactose 3-O-sulfotransferase; D005690:Galactose