Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurenesis lectin binding in a new Chinese hamster ovary cell line

Xiaomei Bai 0 Jillian R. Brown 0 Ajit Varki 0 Jeffrey D. Esko 0 0 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 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 Gal3SO41-4anhydromannitol. 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 Gal1-4(3)GlcNAcO-naphthalenemethanol. - The major sulfated glycans in vertebrates generally consist of glycosaminoglycans, such as chondroitin sulfate and heparan sulfate, which contain hundreds of sulfate residues covalently attached to the chains. Sulfated glycans on glycoproteins have been reported as well, in the form of Man4SO4 (Yamashita et al., 1983), Man6SO4 (Freeze and Wolgast, 1986), GlcNAc6SO4 (Edge and Spiro, 1984; Roux et al., 1988; Spiro 1To whom correspondence should be addressed and Bhoyroo, 1988; de Waard et al., 1991; Noguchi et al., 1992; Sampath et al., 1992; Hokke et al., 1993; Shilatifard et al., 1993; Hemmerich et al., 1994; Hemmerich and Rosen, 1994; Crommie and Rosen, 1995; Lo-Guidice et al., 1995), Gal3SO4 (Spiro and Bhoyroo, 1988; de Waard et al., 1991; Lo-Guidice et al., 1995, 1994; Kamerling et al., 1988; Hard et al., 1992), Gal6SO4 (Hemmerich et al., 1994, 1995; Hemmerich and Rosen, 1994; Brown et al., 1994; Fukuta et al., 1997; Shailubhai et al., 1999), GalNAc4SO4 (Green and Baenziger, 1988a,b; Bergwerff et al., 1995), and GlcA3SO4 (Chou et al., 1986, 1987; Margolis and Margolis, 1993; Schachner and Martini, 1995). Although their relative abundance is low compared to the sulfated glycosaminoglycans, glycans containing these sulfated sugars can have specific and potent biological properties. For example, the recognition of peripheral GalNAc4SO4 residues by receptors on hepatic reticuloendothelial cells facilitates rapid clearance of lutropin and thyrotropin from the circulation (Fiete et al., 1991). The sulfated, sialylated oligosaccharides on O-linked glycoproteins expressed on high endothelial venules facilitate high-affinity binding to L-selectin on leukocytes, thus enabling the rolling and eventual extravasation of the cells into lymph nodes (Vestweber and Blanks, 1999). The potent activities encoded in these glycans depend on specific proteincarbohydrate interactions in which the sulfate group plays an essential role in determining the affinity and specificity of binding. The various sulfation reactions that give rise to the sulfated glycans are catalyzed by distinct sulfotransferases located in the Golgi. Several of the glycoprotein-specific sulfotransferases have been purified and characterized (Kato and Spiro, 1989; Hooper et al., 1995; Spiro et al., 1996; Spiro and Bhoyroo, 1998), and cDNAs for a Gal 6-O-sulfotransferases (Fukuta et al., 1997), GlcNAc 6-sulfotransferases (Uchimura et al., 1998a,b; Lee et al., 1999; Bistrup et al., 1999), and a GlcA 3-O-sulfotransferase (Ong et al., 1998; Bakker et al., 1997) have been cloned. Like other Golgi transferases, all of the enzymes appear to be type II transmembrane glycoproteins and show a high degree of specificity for their oligosaccharide substrates. By analogy to the sulfotransferases involved in glycosaminoglycan assembly, it seems likely that each enzyme is part of a multigene family whose members differ in distribution and developmental expression (Hashimoto et al., 1992; Eriksson et al., 1994; Orellana et al., 1994; Bakker et al., 1997; Honke et al., 1997, Kobayashi et al., 1997, 1999; Habuchi et al., 1998; Ong et al., 1998; Aikawa and Esko, 1999; Shworak et al., 1999; Aikawa et al., 2001). 3-O-sulfation of Gal residues on glycoproteins was first described on thyroglobulins from various species (Spiro and Bhoyroo, 1988; de Waard et al., 1991). The 3-O-sulfotransferase activity responsible for its assembly exhibits selectivity for the terminal disaccharide, Gal1-4GlcNAc-, independently of the underlying oligosaccharide or glycoprotein (Kato and Spiro, 1989). Because 2-3 sialyltransferases, 1-2 fucosyltransferases, and 1-3 Gal transferase act on identical substrates, the four enzymes could potentially compete with each other and convey different chemical and possibly biological properties to the modified chains. Here, we report the activation of a glycoprotein Gal 3-O-sulfotransferase in two Chinese hamster ovary (CHO) cell lines by introduction of a wild-type cDNA library into a strain devoid of glycosaminoglycans (Esko et al., 1985). As expected, sulfation results in decreased sialylation. Surprisingly, a heretofore unappreciated reactivity of Maackia amurensis lectin (MAL) for 3-O-sulfated Gal-terminated oligosaccharides was demonstrated. Results and discussion Enhanced sulfation of macromolecules in clone 26 and 489 In CHO cells, glycosaminoglycans dominate the sulfated oligosaccharide fraction, accounting for >95% of 35SO4 incorporation into all macromolecules. Thus, in the CHO cell mutant, pgsA-745, sulfation is greatly reduced due to a defect in xylosyltransferase, which initiates glycosaminoglycan biosynthesis (Esko et al., 1985). Using this strain, we thought that it might be possible to clone the missing enzyme or for a sulfotransferase that acts on other oligosaccharides by introducing a cDNA library from wild-type CHO cells into the mutant and screening for cells that incorporated normal amounts of 35SO4. To analyze a large number of cells, individual colonies generated from transfected cells were replica plated onto discs of polyester cloth (see Materials and methods). The replicated colonies on the discs were then screened semi-quantitatively by autoradiographic measurement of 35SO4 incorporation into acidprecipitable macromolecules. An occasional colony appear (...truncated)