Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurenesis 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. Pe ptide:N-glycanase F released nearly all of the sulfate label, indicating th at sulfation had occurred selectively on the Asn-linked glycans. Hydrazinol ysis followed by nitrous acid treatment at pH 4 and borohydride reduction y ielded reduced sulfated disaccharides that comigrated with standard Gal3SO( 4)beta1-4anhydromannitol. The disaccharides were resistant to periodate oxi dation but became sensitive after the sulfate group was removed by methanol ysis, indicating that the sulfate was located at C3 of the galactose residu es. Maackia amurensis lectin bound to the sulfated glycopeptides on the cel l surface and in free form, even after sialidase treatment. This finding in dicates that the lectin requires only a charged group at C3 of the galactos e unit and not an intact sialic acid. Growth of cells with chlorate restore d sialidase sensitivity to lectin binding, indicating that sulfation and si alylation occurred largely at the same sites. The enhanced sulfation was du e to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Gal beta1-4(3)GlcNAc beta -O-nap hthalenemethanol.