Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2′-fucosyllactose: Implications in the synthesis of the ligand for L-selectin (original) (raw)
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Glycobiology, 1997
investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Gaipi,3GalNAca-O-Al, Gaipi,4GlcNAcP-O-Al, Gaipi,3GlcNAcp-O-Al, and mucin-type Gaipi,4GlcNAcpi,6(Gaipi,3)GalNAca-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Gaipi, 3GalNAca-linkage and the other (Group B) was directed toward the Gaipi,4GIcNAc branch pl,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Gaip-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward 31,3 linked Gal, particularly, the T-hapten without p 1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galpl,3GalNAca-O-Al and Gaipi,4GIcNAc31,6(Gaipi,3)GalNAca-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg 2 * stimulated Group A 55% and Group B 150%, whereas Mn +2 stimulated Group B 130% but inhibited Group A 75%. Ca 2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-Osulfotransferase activities by fucose, sulfate, and other substitoents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galpl^GalNAca-O-Al to act as an acceptor for Group A. (2) An al,3-fucosyl residue on the pi,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked pi,3 to GalNAca-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the pl,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked pl,3 to GalNAca. (5) Group A activity on Gal linked pi,3 to GalNAc remained unaffected by 3'-sulfation of the pi,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Gaipi,4GlcNAcP-O-Al and Gaipi^GlcNAcP-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5-to 7-fold active, respectively, in their ability to serve as acceptors for Group B.
Journal of Biological Chemistry, 1991
The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAcpl-4GlcNAc@1-2 Mana-. Using a chemically synthesized trisaccharide GalNAc@1-4GlcNAc~l-2Manal-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-0-sulfation of the terminal 6-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3'phosphoadenosine 5'-phosphosulfate ([35S]PAPS). The sulfated product [""SISGGnM-MCO is separated from ["%]PAPS, PAPS degradation products, and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the ["5S]SGGnM-MC0 produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent K , (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 I.LM and for GGnM-MCO of 9 PM. Incorporation of sulfate into the trisaccharide is stimulated 3fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAc-GlcNAc-Man-may modulate GGnM-4-sulfotransferase. The glycoprotein hormones lutropin (LH)' and thyrotropin (TSH) are synthesized in the anterior pituitary and bear unique oligosaccharides with the terminal sequence so,-4GalNAc/31-4GlcNAcpl-2Mana-(1, 2). Reports of other gly-Diabetes and Digestive and Kidney Diseases Grant R01-DK41738 * This investigation was supported in part by National Institute of and by the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part hy the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Glycobiology, 2001
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 Gal3SO 4 β1-4anhydromannitol. 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 Galβ1-4(3)GlcNAcβ-O-naphthalenemethanol.
Journal of Biological Chemistry, 1997
We have isolated a cDNA clone encoding human 3-phosphoadenylylsulfate:galactosylceramide 3-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8-to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells. Sulfoglycolipids are a class of acidic glycolipids containing sulfate esters on their oligosaccharide chains which were originally found in the human brain by Thudichum (1). Sulfoglycolipids are abundant in myelin, spermatozoa, kidney, and small intestine (for review, see Ref. 2) and have been implicated in a variety of physiological functions through their interactions with extracellular matrix proteins, cellular adhesive receptors, blood coagulation systems, complement activation systems, cation transport systems, and microorganisms (for a review, see Ref. 3). The addition of sulfate ester is catalyzed by a sulfotransferase with PAPS 1 serving as the sulfate donor.
Journal of Biological Chemistry, 2002
N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc-6ST) catalyzes the transfer of sulfate from adenosine 3-phosphate,5-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAc1-6[Gal1-3]GalNAc-pNP (core 2), GlcNAc1-6ManOMe, and GlcNAc1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc-6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAc-1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of L-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A.
Biochemistry, 1995
The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated 0-glycan structures was studied by examining the specificities of a2,3-~ialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105,0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Galpl,3GalNAca-unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW -40 OOO) containing -40 T-haptens and asialo Cowper's gland mucin containing -48 T-haptens was -5-fold more active as an acceptor as compared to Galpl, 3GalNAca-0-Al on a molecular weight basis. The second enzyme, a cloned a-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-0-methyl derivatives of the Galpl,3GlcNAcp-unit; C-3 and C-6 substitution on Gal abolished sialylation. G@l,4GlcNAcp-and its 6-sulfo derivative were -20% active; the Lewis a structure, G@1,3-(Fucal,4)GlcNAcp-, was not an acceptor. The acrylamide copolymers containing -40 units of Galp1,-3GlcNAcP-, Gal~l,3(6-~ulfo)GlcNAcp-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to a2,3specific sialidase. A partially purified Colo 205 Lewis type ( a l , 314) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAca2, 3Galp1, 3(6-sulfo)GlcNAc~-O-Ally1 and copolymer containing [9-3H]NeuAca2, 3Galp1, 3(6-sulfo)GlcNAcp-units, using GDP[ 14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal~l,3(6-~ulfo)GalNAcabut was only