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Differential Expression of GalNAc-4-sulfotransferase and GalNAc-transferase Results in Distinct Glycoforms of Carbonic Anhydrase VI in Parotid and Submaxillary Glands (∗)

  • Lora V. Hooper
    Footnotes
    Affiliations
    Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Mary C. Beranek
    Affiliations
    Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Stephen M. Manzella
    Affiliations
    Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Jacques U. Baenziger
    Correspondence
    To whom correspondence and reprint requests should be addressed. Tel.: 314-362-8730; Fax: 314-362-8888
    Affiliations
    Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Author Footnotes
    ∗ This work was supported in part by NIDDK Grant R01-DK41738 (to J. U. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Howard Hughes Medical Institute Predoctoral Fellow.
Open AccessPublished:March 17, 1995DOI:https://doi.org/10.1074/jbc.270.11.5985
      Differential expression of glycosyltransferases has the potential to generate functionally distinct glycoforms of otherwise identical proteins. We have previously demonstrated the presence of unique oligosaccharides terminating with GalNAc-4-SO4 on the pituitary glycoproteins lutropin (LH), thyroid stimulating hormone (TSH), and pro-opiomelanocortin (POMC). A glycoprotein hormone:GalNAc-transferase and a GalNAc-4-sulfotransferase are present in the pituitary and can account for the synthesis of these unique oligosaccharides on specific glycoproteins. Both transferases are coordinately expressed in a number of tissues in addition to pituitary, including submaxillary gland, lacrimal gland, and kidney, suggesting that additional glycoproteins bearing oligosaccharides terminating with GalNAc-4-SO4 are synthesized in these tissues. In this study we show that while the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase are coordinately expressed in bovine submaxillary gland, the GalNAc-transferase is expressed in the parotid gland in the absence of the GalNAc-4-sulfotransferase. The relative expression of these two transferases in submaxillary and parotid glands correlates with the presence of unique Asn-linked oligosaccharides on carbonic anhydrase VI (CA VI) synthesized in each of these tissues. The majority of Asn-linked oligosaccharides on CA VI synthesized in submaxillary gland terminate with GalNAc-4-SO4. In contrast, CA VI which is synthesized in bovine parotid gland bears oligosaccharides which terminate predominantly with β1,4-linked GalNAc which is not sulfated. The presence of different terminal residues on the Asn-linked oligosaccharides of submaxillary and parotid CA VI thus correlates with the complement of transferases in these glands and suggests differing biological roles for submaxillary and parotid CA VI.

      INTRODUCTION

      Glycoproteins expressed in cells which differ in their complement of glycosyltransferases will potentially bear oligosaccharides differing in structure, thus producing distinct glycoforms of otherwise identical proteins which may differ in their biologic functions. We demonstrated this to be the case for equine lutropin (LH) (
      The abbreviations used are: LH
      luteinizing hormone
      TSH
      thyroid stimulating hormone
      POMC
      proopiomelanocortin
      TFPI
      tissue factor pathway inhibitor
      CA
      carbonic anhydrase
      GalNAc
      N-acetylgalactosamine
      GlcNAc
      N-acetylglucosamine
      Man
      mannose
      Gal
      galactose
      Sia
      sialic acid
      Asn
      asparagine
      PAPS
      adenosine 3′-phosphate 5′-phosphosulfate
      GGnM-MCO
      GalNAcβ1,4GlcNAcβ1,2Manα1-O(CH2)8COOCH3
      PNGase F
      peptide:N-glycosidase F
      HPLC
      high performance liquid chromatography
      ConA
      concanavalin A
      WFA
      W. floribunda agglutinin.
      )and chorionic gonadotropin (CG) which are synthesized in the anterior lobe of the pituitary and the placenta, respectively(
      • Smith P.L.
      • Bousfield G.R.
      • Kumar S.
      • Fiete D.
      • Baenziger J.U.
      ). Equine LH and CG bear Asn-linked oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα and sialic acid α2,3Galβ1,4GlcNAcβ1,2Manα, respectively, because of differences in the complement of glycosyltransferases expressed in pituitary and placenta. We had previously demonstrated that the pituitary glycoprotein hormones LH and thyrotropin (TSH) from a number of different mammalian species bear Asn-linked oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα (
      • Green E.D.
      • Van Halbeek H.
      • Boime I.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      • Green E.D.
      ,
      • Stockell Hartree A.
      • Renwick A.G.C.
      ). We subsequently determined that the sulfated oligosaccharides play an important role following release of LH into the blood. Hepatic reticuloendothelial cells contain a receptor specific for oligosaccharides terminating with GalNAc-4-SO4(
      • Fiete D.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ), which mediates the rapid removal of LH from the circulation (
      • Baenziger J.U.
      • Kumar S.
      • Brodbeck R.M.
      • Smith P.L.
      • Beranek M.C.
      ) thus regulating its circulatory half-life.
      Glycoprotein hormone oligosaccharides terminating with β1,4-linked GalNAc-4-SO4 are synthesized by the sequential action of two highly specific enzymes. GalNAc is added to the synthetic intermediate GlcNAc2Man3GlcNAc2Asn by a protein-specific GalNAc-transferase(
      • Smith P.L.
      • Baenziger J.U.
      ). In the presence of a specific protein recognition motif, the Km for GalNAc addition to this synthetic intermediate by the glycoprotein hormone:GalNAc-transferase is 5-10 μM, in contrast to a Km of 1-2 mM for addition to the same oligosaccharide intermediate in the absence of the recognition marker (
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Smith P.L.
      • Baenziger J.U.
      ). The specificity of the transferase accounts for the addition of GalNAc to LH and TSH but not to other pituitary glycoproteins. Addition of sulfate to the 4-hydroxyl of terminal GalNAc residues occurs by the action of a GalNAc-4-sulfotransferase which is not proteinspecific(
      • Green E.D.
      • Gruenebaum J.
      • Bielinska M.
      • Baenziger J.U.
      • Boime I.
      ,
      • Green E.D.
      • Morishima C.
      • Boime I.
      • Baenziger J.U.
      ,
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ), requiring only the terminal sequence GalNAcβ1,4GlcNAcβ for the transfer of sulfate from adenosine 3′-phosphate 5′-phosphosulfate (PAPS). (
      L. V. Hooper and J. U. Baenziger, unpublished observation.
      )The absence of GalNAc-4-SO4 on equine and human CG is accounted for by the lack of expression of either of these transferases in equine and human placenta.
      Oligosaccharides terminating with GalNAc-4-SO4 have to date been shown to be major constituents on two glycoproteins which are not members of the glycoprotein hormone family: pro-opiomelanocortin (POMC) (
      • Skelton T.P.
      • Kumar S.
      • Smith P.L.
      • Beranek M.C.
      • Baenziger J.U.
      ,
      • Siciliano R.A.
      • Morris H.R.
      • McDowell R.A.
      • Azadi P.
      • Rogers M.E.
      • Bennett H.P.
      • Dell A.
      ,
      • Siciliano R.A.
      • Morris H.R.
      • Bennett H.P.J.
      • Dell A.
      ) and recombinant tissue factor pathway inhibitor (TFPI)(
      • Smith P.L.
      • Skelton T.P.
      • Fiete D.
      • Dharmesh S.M.
      • Beranek M.C.
      • MacPhail L.
      • Broze Jr., G.J.
      • Baenziger J.U.
      ). POMC synthesized by AtT-20 cells, a pituitary corticotroph-derived cell line(
      • Skelton T.P.
      • Kumar S.
      • Smith P.L.
      • Beranek M.C.
      • Baenziger J.U.
      ), and the 16-kDa amino-terminal fragment derived from bovine POMC (
      • Siciliano R.A.
      • Morris H.R.
      • Bennett H.P.J.
      • Dell A.
      ,
      • Verostek M.F.
      • Atkinson P.H.
      • Trimble R.B.
      ) both have oligosaccharides terminating with GalNAc-4-SO4. TFPI was known to bear sulfated oligosaccharides when synthesized by endothelial cells (
      • Colburn P.
      • Buonassisi V.
      ,
      • Warn-Cramer B.J.
      • Maki S.L.
      • Rapaport S.I.
      ) and was predicted to be a substrate for the glycoprotein hormone:GalNAc-transferase based on the presence of a putative GalNAc-transferase recognition sequence. The Asn-linked oligosaccharides on recombinant TFPI synthesized in 293 cells, which express both the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase, were subsequently shown to consist almost exclusively of structures terminating with GalNAc-4-SO4(
      • Smith P.L.
      • Skelton T.P.
      • Fiete D.
      • Dharmesh S.M.
      • Beranek M.C.
      • MacPhail L.
      • Broze Jr., G.J.
      • Baenziger J.U.
      ).
      We first identified the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase in extracts prepared from pituitary, the site of LH and TSH synthesis(
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Gruenebaum J.
      • Bielinska M.
      • Baenziger J.U.
      • Boime I.
      ,
      • Green E.D.
      • Morishima C.
      • Boime I.
      • Baenziger J.U.
      ,
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ). Both enzymes were subsequently localized to several other tissues including submaxillary gland, kidney, and lacrimal gland but were absent in a number of the tissues surveyed, such as heart and liver(
      • Dharmesh S.M.
      • Skelton T.P.
      • Baenziger J.U.
      ). Expression of the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase in tissues other than pituitary suggests that glycoproteins bearing oligosaccharides terminating with GalNAc-4-SO4 are synthesized in these tissues; however, glycoproteins bearing such structures as major components have not previously been identified.
      We report here that both the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase are expressed in bovine submaxillary gland and that the majority of Asn-linked oligosaccharides present on the secreted form of carbonic anhydrase (CA VI) synthesized by bovine submaxillary gland terminate with GalNAc-4-SO4. In addition, we demonstrate that a similar proportion of Asn-linked oligosaccharides on CA VI synthesized in the bovine parotid gland terminate with β1,4-linked GalNAc which is not sulfated. The latter structure is consistent with our determination that the glycoprotein hormone:GalNAc-transferase but not the GalNAc-4-sulfotransferase is expressed in the parotid gland. Oligosaccharides terminating with GalNAc-4-SO4 have been described on Tamm-Horsfall glycoprotein as minor components(
      • Hård K.
      • Van Zadelhoff G.
      • Moonen P.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      ); however, this is the first instance of a non-pituitary glycoprotein in which oligosaccharides terminating with GalNAc-4-SO4 represent the major component. Our observations suggest that a crucial function exists for the Asn-linked oligosaccharides on CA VI, and, furthermore, that submaxillary and parotid CA VI may have distinct biological roles based on the differences in the structures of their Asn-linked oligosaccharides.

      EXPERIMENTAL PROCEDURES

      Materials

      Bovine tissues were obtained from Pel Freez. [35S]PAPS was enzymatically synthesized as described previously (
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ) using [35S]SO4 from ICN. The synthesis of GGnM-MCO has been published previously(
      • Vandana Hindsgaul O.
      • Baenziger J.U.
      ,
      • Srivastava V.
      • Hindsgaul O.
      ). Peptide:N-glycosidase F (PNGase F) (
      • Plummer Jr., T.H.
      • Elder J.H.
      • Alexander S.
      • Phelan A.W.
      • Tarentino A.L.
      ,
      • Tarentino A.L.
      • Gómez C.M.
      • Plummer T.H.
      ) and Newcastle disease virus neuraminidase (
      • Paulson J.C.
      • Weinstein J.
      • Dorland L.
      • Van Halbeek H.
      • Vliegenthart J.F.G.
      ) were purified as described. Wisteria floribunda agglutinin (WFA) and WFA-agarose were obtained from E-Y Laboratories.

      Assay of Glycoprotein Hormone:GalNAc-transferase and GalNAc-4-sulfotransferase Activities

      Total membrane fractions were prepared by mincing and homogenizing bovine submaxillary, parotid, or thyroid glands in 15 mM HEPES (pH 7.4), 1 mM EDTA (5 ml/g of tissue) using a Polytron (Brinkmann Instruments). The extracts were sedimented at 10,000 × g for 15 min, and the supernatants then sedimented at 100,000 × g for 1 h. The pellets were washed with 15 mM HEPES (pH 7.4) and suspended in 15 mM HEPES (pH 7.4), 13% glycerol, and 0.1% Triton X-100. Protein was determined by the Bradford dye-binding assay (Bio-Rad) using bovine γ-globulin as standard.
      Glycoprotein hormone:GalNAc-transferase assays were incubated at 37°C for 90 min. Each assay of 50 μl contained 25 mM HEPES (pH 7.5), 0.1% Triton X-100, 10 mM ATP, 1 mg/ml BSA, 15% glycerol, 10 mM MnCl2, protease inhibitors, 1 mM UDP-GalNAc, 165 ng of agalacto-hCG as acceptor substrate, and tissue extract. GalNAc incorporation into hCG was quantitated using biotinylated Wisteria floribunda agglutinin as described(
      • Mengeling B.J.
      • Smith P.L.
      • Stults N.L.
      • Smith D.F.
      • Baenziger J.U.
      ).
      GalNAc-4-sulfotransferase reactions (50 μl) were carried out as described (
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ) at 28°C for 2 h and contained 15 mM HEPES (pH 7.4), 1% Triton X-100, 40 mM β-mercaptoethanol, 10 mM NaF, 1 mM ATP, 4 mM magnesium acetate, 13% glycerol, protease inhibitors, 2 μM unlabeled PAPS, 1 × 106 cpm [35S]PAPS, 20 μM GGnM-MCO, and tissue extract. [35S]SO4-GGnM-MCO was separated from [35S]PAPS and from labeled endogenous acceptors by passage over a Sep-Pak (C18) cartridge (Waters) as described previously(
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ). For each tissue extract, a control reaction was done in the absence of GGnM-MCO.

      In Vitro Labeling with GalNAc-4-sulfotransferase

      Each reaction contained the same components as that described above for the GalNAc-4-sulfotransferase assay, except GGnM-MCO and unlabeled PAPS were omitted. Partially purified bovine submaxillary gland GalNAc-4-sulfotransferase or 1 μg of purified CA VI and 5 μl of partially purified bovine pituitary GalNAc-4-sulfotransferase were included in the 50-μl reactions, which were incubated overnight at 28°C. Duplicate reactions were either stopped by addition of an equal volume of sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.003% bromphenol blue, and 62.5 mM Tris, pH 6.8) or were digested with 34 microunits of peptide:N-glycosidase as described (
      • Smith P.L.
      • Bousfield G.R.
      • Kumar S.
      • Fiete D.
      • Baenziger J.U.
      ) followed by the addition of sample buffer.

      Protein Sequence Determination

      A partially purified bovine submaxillary gland GalNAc-4-sulfotransferase preparation was dialyzed extensively against distilled water, lyophilized, and resuspended in SDS-PAGE sample buffer. After boiling for 5 min, the sample was electrophoresed in a 7.5% SDS-polyacrylamide gel, the 45-kDa band was excised from the gel, and the protein was electroeluted in a Bio-Rad electroelution chamber in 50 mM NH4HCO3, 0.1% SDS. The eluate was dialyzed overnight against 50 mM NH4HCO3, lyophilized, and extracted in acetone-triethylamine-acetic acid-water (86:5:5:4) to remove any residual SDS. The precipitate was washed in acetone and resuspended in water. 75 μg of the purified protein was digested with sequencing grade trypsin or chymotrypsin (Boehringer Mannheim) overnight at 37°C. Individual peptides were purified by reverse-phase capillary HPLC and were subjected to amino acid sequence analysis by automated Edman degradation. The resulting sequences were used to search the NBRF and Swiss protein data bases.

      Purification of Carbonic Anhydrase VI

      Carbonic anhydrase VI was purified from bovine submaxillary or parotid glands essentially according to the method of Fernley et al.(
      • Fernley R.T.
      • Coghlan J.P.
      • Wright R.D.
      ). 200 g of frozen glands were put through a meat grinder and homogenized in 1 liter of 50 mM NaPO4 (pH 7.4), 1 mM EDTA. The homogenate was centrifuged at 8000 × g, and the supernatant was filtered through cheesecloth and then precipitated with an equal volume of a saturated ammonium sulfate solution for 1 h. The solution was centrifuged at 10,000 × g, and the precipitate was resuspended in 2 volumes of 0.1 M NH4HCO3 and dialyzed overnight against 4 liters of 0.1 M NH4HCO3 with one buffer change. After centrifuging the dialysate at 10,000 × g, the solution was passed over 25 ml of p-aminomethylbenzene sulfonamide-agarose (Sigma) followed by washing with 750 ml each of 0.1 M NH4HCO3 and 0.2 M NaI in 0.1 M NH4HCO3. The column was eluted in 200 ml of 0.4 M NaN3 in 0.1 M NH4HCO3, and the fractions containing protein were pooled and dialyzed overnight at 4°C against 25 mM Tris, pH 7.4. The dialysate was recovered and bound to a 15-ml DEAE-Sepharose column equilibrated in 25 mM Tris, pH 7.4, followed by washing in 200 ml of 50 mM NaCl, 25 mM Tris (pH 7.4) and elution in 100 ml of 200 mM NaCl, 25 mM Tris, pH 7.4. Fractions containing protein were pooled and tested for carbonic anhydrase activity in the phenol red assay described by Sundaram et al.(
      • Sundaram V.
      • Rumbolo P.
      • Grubb J.
      • Strisciuglio P.
      • Sly W.
      ).

      Lectin Affinity Chromatography

      Oligosaccharides were separated into bound and unbound fractions on concanavalin A-Sepharose (Pharmacia Biotech Inc.) as described(
      • Smith P.L.
      • Bousfield G.R.
      • Kumar S.
      • Fiete D.
      • Baenziger J.U.
      ). Oligosaccharides containing terminal β1,4-linked GalNAc were isolated on W. floribunda agglutinin-agarose columns as described(
      • Smith P.L.
      • Bousfield G.R.
      • Kumar S.
      • Fiete D.
      • Baenziger J.U.
      ).

      Western Blot

      Anti-GalNAc-4-SO4 monoclonal antibody was biotinylated with biotin-LC-hydrazide (Pierce) according to the manufacturer's instructions. W. floribunda agglutinin was biotinylated as described(
      • Mengeling B.J.
      • Smith P.L.
      • Stults N.L.
      • Smith D.F.
      • Baenziger J.U.
      ). CA VI purified from bovine submaxillary and parotid glands was subjected to SDS-PAGE in a 10% gel and electroblotted onto Immobilon-P membranes (Millipore) which were blocked in 5% powdered milk in phosphate-buffered saline, 0.5% Tween 20 and incubated with 2 μg/ml biotinylated anti-GalNAc-4-SO4 monoclonal antibody or with 40 ng/ml biotinylated W. floribunda agglutinin (WFA) for 1 h. After washing in phosphate-buffered saline, 0.5% Tween 20 blots were incubated with streptavidin-peroxidase (Sigma) at 400 ng/ml for 1 h, detected with chemiluminescence reagent (DuPont NEN), and exposed to film.

      Enzymatic Digestions

      Peptide:N-glycosidase digestions were carried out as described(
      • Smith P.L.
      • Bousfield G.R.
      • Kumar S.
      • Fiete D.
      • Baenziger J.U.
      ). Digestions with Newcastle disease virus neuraminidase and diplococcal β-hexosaminidase were performed in 50 mM sodium cacodylate at pH 6.0. Digestion with jack bean β-hexosaminidase was carried out in 50 mM citrate at pH 4.5. Digestions with human recombinant GalNAc-4-sulfatase (
      • Litjens T.
      • Morris C.P.
      • Gibson G.J.
      • Beckmann K.R.
      • Hopwood J.J.
      ,
      • Anson D.S.
      • Muller V.
      • Bielicki J.
      • Harper G.S.
      • Hopwood J.J.
      ), generously provided by Dr. J. J. Hopwood (Adelaide Children's Hospital, Australia), were performed in 50 mM sodium acetate buffer, pH 5.8, containing 0.1 mg/ml bovine serum albumin, 10 mM MnCl2 for oligosaccharides and in the same buffer containing a final concentration of 20 μg/ml of each of the following protease inhibitors: aprotinin, leupeptin, antipain, pepstatin, and chymostatin for glycoproteins.

      High Performance Liquid Chromatography (HPLC)

      Asn-linked oligosaccharides labeled in vitro with [35S]SO4 from [35S]PAPS were released by peptide:N-glycosidase F digestion, purified by passage over a Sep-Pak C18 cartridge, and desalted by passage over Bio-Gel P-2 (Bio-Rad) in water. The purified oligosaccharides were fractionated by anion exchange chromatography on a Glycopak HPLC column (Waters) with elution in a linear gradient of 0-100 mM NaCl in deionized water over 60 min at a flow rate of 0.5 ml/min. Elution positions of sulfated and sialylated oligosaccharides were determined using previously characterized authentic standards(
      • Green E.D.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ). Purified [35S]SO4-labeled oligosaccharides were analyzed for sulfate esters by treatment with 40 mM HCl at 100°C for 2 h to release the sulfated monosaccharides which were separated from incompletely hydrolyzed oligosaccharides and from free [35S]SO4 by passage over a Sephadex G-10 column. The sulfated monosaccharides were analyzed on a CarboPak PA-1 column (Dionex) as described(
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ). Authentic standards were used to determine the elution positions of GalNAc and GlcNAc sulfated esters.
      For monosaccharide composition analysis, 40 μg of purified submaxillary or parotid CA VI was hydrolyzed at 100°C in 4 N HCl for 4 h. The hydrolysates were dried down under vacuum and resuspended in distilled water, and the monosaccharides were separated isocratically on a CarboPak PA1 column in 16 mM NaOH at a flow rate of 0.5 ml/min. The column effluent was mixed with an equal volume of 300 mM NaOH before entering a pulsed amperimetric detection cell. Elution positions of monosaccharides were determined by comparison with authentic standards.
      For analysis by anion exchange HPLC and by ion suppression amine adsorption, Asn-linked oligosaccharides were released from purified bovine submaxillary and parotid carbonic anhydrase VI by digestion with peptide:N-glycosidase F, purified by passage over a Sep-Pak C18 cartridge in water, and desalted by passage over a Bio-Gel P-2 column (Bio-Rad). The oligosaccharides were labeled at their reducing termini by reduction with [3H]NaBH4 (American Radiochemical) as described previously(
      • Green E.D.
      • Baenziger J.U.
      ). The 3H-labeled oligosaccharides were fractionated by anion exchange chromatography on MicroPak AX-5 (Varian Associates) at pH 4.0 using a gradient of KH2PO4 as described previously(
      • Green E.D.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      ). Ion suppression amine adsorption HPLC on MicroPak AX-5 (Varian Associates) was carried out as described previously(
      • Baenziger J.U.
      ,
      • Mellis S.J.
      • Baenziger J.U.
      ).

      RESULTS

      Relative Expression of Glycoprotein Hormone:GalNAc-transferase and GalNAc-4-sulfotransferase Differs in Bovine Submaxillary and Parotid Glands

      We have previously shown that a GalNAc-transferase and GalNAc-4-sulfotransferase with the same specificity and properties as the glycoprotein hormone:GalNAc-transferase and GalNAc-4-sulfotransferase found in pituitary are expressed at high levels in a number of rat tissues, including the submaxillary gland, lacrimal gland, and kidney(
      • Dharmesh S.M.
      • Skelton T.P.
      • Baenziger J.U.
      ). However, endogenous glycoproteins bearing oligosaccharides terminating with β1,4-linked GalNAc-4-SO4 had not been identified in these tissues. The high levels of GalNAc-transferase and GalNAc-4-sulfotransferase expression seen in the rat submaxillary gland suggested it would be an excellent tissue source for purification if similar levels were expressed in bovine glands. We therefore analyzed crude membrane fractions from bovine submaxillary, parotid, and thyroid glands for the presence of GalNAc-transferase and GalNAc-4-sulfotransferase activities.
      High levels of glycoprotein hormone:GalNAc-transferase activity are detected in bovine submaxillary and parotid glands (Table 1). In contrast, GalNAc-4-sulfotransferase levels which are significantly higher than those in pituitary extracts are detected in submaxillary gland extracts, whereas little or no activity is detected in bovine parotid gland extracts (Table 1). Little or no activity for either transferase is detected in bovine thyroid gland, in agreement with previous results in rat tissue (
      • Dharmesh S.M.
      • Skelton T.P.
      • Baenziger J.U.
      ). The high levels of glycoprotein hormone:GalNAc-transferase and GalNAc-4-sulfotransferase in submaxillary gland suggest the presence of endogenous glycoproteins bearing β1,4-linked GalNAc-4-SO4. Likewise, the presence of GalNAc-transferase but not GalNAc-4-sulfotransferase activity in parotid gland extracts makes it likely that this gland synthesizes glycoproteins which terminate in β1,4-linked GalNAc. The high levels of transferase activity in bovine submaxillary glands make it an excellent tissue source for purification of the GalNAc-4-sulfotransferase.

      Carbonic Anhydrase VI Is an Endogenous Substrate for GalNAc-4-sulfotransferase

      During the course of purification of GalNAc-4-sulfotransferase from bovine submaxillary glands, we observed that [35S]SO4 was incorporated into an endogenous 45-kDa acceptor when partially purified GalNAc-4-sulfotransferase was incubated with [35S]PAPS in the absence of any exogenous acceptor (Fig. 1, lanes 1 and 2). The incorporated [35S]SO4 was completely released from the 45-kDa protein upon digestion with PNGase F, indicating that the incorporated SO4 is present exclusively on Asn-linked oligosaccharides (Fig. 1, lane 3). This raised the possibility that the 45-kDa protein might be a major endogenous acceptor for the GalNAc-4-sulfotransferase in the submaxillary gland.
      Figure thumbnail gr1
      Figure 1An endogenous 45-kDa glycoprotein from bovine submaxillary gland incorporates [35S]SO4 into its Asn-linked oligosaccharides. A partially purified preparation of GalNAc-4-sulfotransferase from bovine submaxillary glands was incubated with [35S]PAPS as outlined under “Experimental Procedures.” Duplicate reactions were either not digested (lane 1), mock-digested (lane 2), or digested with PNGase F (lane 3) and analyzed by SDS-PAGE (10%) and autoradiography. The 45-kDa species is indicated with an arrow.
      Sulfate incorporation into Asn-linked oligosaccharides has been described in at least five different linkages (Man-6-SO4, Man-4-SO4, GalNAc-4-SO4, Gal-3-SO4, and GlcNAc-6-SO4)(
      • Freeze H.H.
      • Wolgast D.
      ,
      • Yamashita K.
      • Ueda I.
      • Kobata A.
      ,
      • Green E.D.
      • Boime I.
      • Baenziger J.U.
      ,
      • Spiro R.G.
      • Bhoyroo V.D.
      ,
      • Roux L.
      • Holojda S.
      • Sundblad G.
      • Freeze H.H.
      • Varki A.
      ). Since the GalNAc-4-sulfotransferase preparation used in the in vitro labeling reaction was only partially purified, we determined the location and linkage of the sulfate on the Asn-linked oligosaccharides of the endogenous acceptor. The [35S]SO4-labeled 45-kDa protein was separated from other endogenous sulfate acceptors by SDS-PAGE, eluted from the polyacrylamide gel, and treated with PNGase F. The labeled oligosaccharides were fractionated on concanavalin A (ConA)-Sepharose into bound (51% of the incorporated counts) and unbound (49% of the incorporated counts) fractions. The ConA-Sepharose bound and unbound oligosaccharides were each subjected to mild acid hydrolysis under conditions that cleave glycosidic bonds more rapidly than sulfate esters(
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ). The labeled monosaccharides were separated from incompletely hydrolyzed oligosaccharides and from free [35S]SO4 by chromatography on Sephadex G-10 and were analyzed by HPLC on a CarboPak PA1 column (Dionex). The only sulfated monosaccharide product detected in the ConA-bound (Fig. 2) and unbound (data not shown) fractions co-migrated with authentic GalNAc-4-SO4. This suggested that at least a fraction of the 45-kDa glycoprotein bears Asn-linked oligosaccharides terminating with β1,4-linked GalNAc which are subject to sulfation by the GalNAc-4-sulfotransferase which transfers sulfate exclusively to structures terminating in GalNAc in a β1,4 linkage(
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ).
      Figure thumbnail gr2
      Figure 2The [35S]SO4-labeled Asn-linked oligosaccharides released from the 45-kDa endogenous substrate in submaxillary glands contain GalNAc-4-SO4. The Asn-linked oligosaccharides from bovine submaxillary 45-kDa glycoprotein were labeled with [35S]SO4 as in . The labeled protein was subjected to SDS-PAGE, electroeluted, and treated with PNGase F. Released Asn-linked oligosaccharides were purified and passed over a ConA-Sepharose column. The ConA-Sepharose-bound fraction was subjected to mild acid hydrolysis and analyzed by HPLC on a CarboPak PA1 column (Dionex). Elution positions for authentic standards are shown.
      Since this 45-kDa glycoprotein was relatively abundant in preparations of partially purified GalNAc-4-sulfotransferase, we purified sufficient material for amino acid sequence determination by preparative separation on SDS-PAGE followed by elution of the 45-kDa band from the gel. Amino-terminal sequence could not be obtained due to blockage of the amino terminus; therefore, internal sequences were obtained on fragments generated by digestion of the protein with chymotrypsin or trypsin. These sequences were used to search the Swiss protein data base and were found to be homologous to sequences within sheep and human CA VI. This suggested that the 45-kDa endogenous substrate in submaxillary gland is CA VI, originally identified in rat (
      • Feldstein J.B.
      • Silverman D.N.
      ) and human (
      • Murakami H.
      • Sly W.S.
      ) saliva as a glycosylated form of carbonic anhydrase which is thought to function in the pH regulation of saliva.
      Sulfonamides are specific inhibitors of carbonic anhydrases which have been used extensively in the purification of a number of these enzymes (
      • Fernley R.T.
      • Coghlan J.P.
      • Wright R.D.
      ,
      • Feldstein J.B.
      • Silverman D.N.
      ,
      • Murakami H.
      • Sly W.S.
      ). Passage of partially purified GalNAc-4-sulfotransferase over p-aminomethylbenzene sulfonamide-agarose resulted in the removal of the 45-kDa endogenous substrate, further supporting the identity of the endogenous substrate as CA VI (not shown). We therefore characterized the Asn-linked oligosaccharides present on CA VI which had been purified from bovine submaxillary and parotid glands by affinity chromatography on p-aminomethylbenzene sulfonamide-agarose using a modification of the procedure of Fernley et al.(
      • Fernley R.T.
      • Coghlan J.P.
      • Wright R.D.
      ). Purified CA VI from both tissues migrated at 45 kDa by SDS-PAGE, which was reduced to 35 kDa by digestion with PNGase F suggesting the presence of two Asn-linked oligosaccharides (Fig. 3). This is consistent with the molecular weights and number of Asn-linked oligosaccharides observed on CA VI isolated from sheep, rat, and human(
      • Fernley R.T.
      • Coghlan J.P.
      • Wright R.D.
      ,
      • Feldstein J.B.
      • Silverman D.N.
      ,
      • Murakami H.
      • Sly W.S.
      ). The specific activity of CA VI purified from bovine submaxillary and parotid glands, as measured by a phenol red pH indicator assay(
      • Sundaram V.
      • Rumbolo P.
      • Grubb J.
      • Strisciuglio P.
      • Sly W.
      ), was 1933 units/mg and 1590 units/mg, respectively, which is similar to that reported for homogeneous sheep CA VI(
      • Fernley R.T.
      • Coghlan J.P.
      • Wright R.D.
      ).
      Figure thumbnail gr3
      Figure 3Purification of carbonic anhydrase VI from bovine submaxillary and parotid salivary glands. CA VI was purified from bovine submaxillary and parotid glands by affinity chromatography on p-aminomethylbenzene sulfonamide-agarose and elution in 0.4 M NaN3 as indicated under “Experimental Procedures.” 2 μg of purified protein from each source was subjected to SDS-PAGE (10%), before(-) and after (+) treatment with PNGase F. Proteins were visualized by staining with Coomassie Blue.
      [35S]SO4 was transferred from [35S]PAPS to both submaxillary and parotid CA VI in vitro by GalNAc-4-sulfotransferase which had been partially purified from bovine pituitaries and was itself free of the 45-kDa endogenous substrate (Fig. 4, lanes 2, 3, 5, and 6). The incorporated [35S]SO4 was quantitatively released from both tissue forms by digestion with PNGase F, indicating that the sulfate was transferred exclusively to the Asn-linked oligosaccharides (Fig. 4, lanes 4 and 7). [35S]SO4-labeled submaxillary CA VI was purified on p-aminomethylbenzene sulfonamide-agarose, digested with PNGase F, and the purified Asn-linked oligosaccharides were subjected to mild acid hydrolysis and analysis on a CarboPak PA1 column as before. A single peak was obtained which co-migrated with authentic GalNAc-4-SO4, indicating that [35S]SO4 was incorporated into CA VI Asn-linked oligosaccharides as GalNAc-4-SO4 (data not shown). Since both submaxillary and parotid CA VI are in vitro substrates for GalNAc-4-sulfotransferase, some fraction of the Asn-linked oligosaccharides from CA VI from both tissues must contain Asn-linked oligosaccharides with terminal β1,4-linked GalNAc.
      Figure thumbnail gr4
      Figure 4The Asn-linked oligosaccharides on purified submaxillary and parotid carbonic anhydrase VI incorporate [35S]SO4. 1 μg of purified CA VI from bovine submaxillary (lanes 2, 3, and 4) and parotid (lanes 5, 6, and 7) glands was incubated with [35S]PAPS and a partially purified GalNAc-4-sulfotransferase preparation from bovine pituitary as outlined under “Experimental Procedures.” Identical reactions were analyzed by SDS-PAGE (10%) and autoradiography either after no digestion (lanes 2 and 5), after mock digestion (lanes 3 and 6), or after digestion with PNGase F (lanes 4 and 7). The reaction in lane 1 contains no exogenous CA VI.
      CA VI isolated from submaxillary and parotid glands, respectively, have nearly identical monosaccharide compositions (Table 2). There is sufficient mannose to account for two complex Asn-linked oligosaccharides with three mannose residues each. CA VI from both glands contains GalNAc as well as Gal, indicating that there are Asn-linked oligosaccharides terminating with GalNAc and/or that there are O-glycosidically linked oligosaccharides as well as Asn-linked oligosaccharides present. If there are no O-glycosidically linked structures present, the amounts of GalNAc present would indicate that a majority of the Asn-linked oligosaccharides present on both submaxillary and parotid CA VI bear one or more GalNAc moieties.

      The Asn-linked Oligosaccharides on CA VI from Bovine Submaxillary Bear Terminal GalNAc-4-SO4 While Those on Parotid CA VI Bear Terminal GalNAc

      The experiments described above indicated that terminal β1,4-linked GalNAc was present on the oligosaccharides of CA VI from bovine submaxillary and parotid gland, allowing CA VI from both tissues to act as an in vitro substrate for the GalNAc-4-sulfotransferase. Since GalNAc-4-sulfotransferase activity was detected in extracts of submaxillary glands but not parotid glands, we expected submaxillary and parotid CA VI to differ in their content of GalNAc-4-SO4. This possibility was addressed by probing a Western blot of CA VI from both tissues with a monoclonal antibody specific for terminal GalNAc-4-SO4(
      • Skelton T.P.
      • Kumar S.
      • Smith P.L.
      • Beranek M.C.
      • Baenziger J.U.
      ,
      • Dharmesh S.M.
      • Baenziger J.U.
      ) and with W. floribunda agglutinin (WFA) which binds specifically to terminal β1,4-linked GalNAc(
      • Nyame K.
      • Smith D.F.
      • Damian R.T.
      • Cummings R.D.
      ). CA VI from submaxillary glands is detected by the anti-GalNAc-4-SO4 monoclonal antibody, indicating the presence of Asn-linked oligosaccharides terminating with GalNAc-4-SO4. In contrast, CA VI from parotid glands does not react with this antibody even at levels 10-fold greater than are required for detection of the sulfated oligosaccharides on submaxillary CA VI (Fig. 5). Submaxillary and parotid CA VI are both detected by W. floribunda agglutinin (Fig. 5). The reactivity with W. floribunda agglutinin and the ability to act as an in vitro substrate for the GalNAc-4-sulfotransferase indicate that a significant proportion of the Asn-linked oligosaccharides on both submaxillary and parotid CA VI terminates with β1,4-linked GalNAc.
      Figure thumbnail gr5
      Figure 5Western blot analyses of submaxillary and parotid carbonic anhydrase VI using an anti-GalNAc-4-SO4 monoclonal antibody and W. floribunda agglutinin. The indicated amounts of affinity-purified submaxillary and parotid CA VI were subjected to SDS-PAGE (10%) followed by electroblotting onto polyvinylidene difluoride. Blots were probed with biotinylated anti-GalNAc-4-SO4 monoclonal antibody or with biotinylated W. floribunda agglutinin (WFA), which specifically recognizes terminal β1,4-linked GalNAc residues. Blots were detected with streptavidin-peroxidase and chemiluminescence.
      The structures of the Asn-linked oligosaccharides from submaxillary and parotid CA VI were examined in greater detail to determine more precisely the proportion of oligosaccharides terminating with GalNAc and GalNAc-4-SO4, as well as other structural features. Asn-linked oligosaccharides were released from CA VI from both tissues by digestion with PNGase F and were labeled at their reducing termini by reduction with [3H]borohydride. When fractionated by anion exchange HPLC, oligosaccharides from the two tissue forms of CA VI displayed markedly different distributions of anionic species (Fig. 6, A and B) with oligosaccharides from submaxillary CA VI yielding a more complex pattern than those from parotid CA VI. Oligosaccharides from submaxillary CA VI co-migrated with authentic standards containing no anionic species(N0), 1 sialic acid(N1), 1 sulfate (S1), 2 sialic acids(N2), 1 sulfate and 1 sialic acid (SN), 2 sulfates (S2), 3 sialic acids(N3), 1 sulfate and 2 sialic acids (S1N2), 2 sulfates and 1 sialic acid (S2N1), and 4 anionic moieties (SxNy where x + y = 4) (Fig. 6A). Treatment with 2 N acetic acid at 100°C for 15 min will release sialic acid but not sulfate from oligosaccharides(
      • Green E.D.
      • Van Halbeek H.
      • Boime I.
      • Baenziger J.U.
      ). Following treatment with 2 N acetic acid, the proportion of oligosaccharides from submaxillary gland CA VI which migrated as neutral species increased from 13% to 44% of the total. Those oligosaccharides not migrating as neutral species after treatment with 2 N acetic acid migrated as oligosaccharides with 1 sulfate (35% migrate as S1) or with 2 sulfate (17% migrate as S2) moieties (not shown). Digestion of the oligosaccharides from submaxillary CA VI with Newcastle disease virus neuraminidase, which will release sialic acid in an α2,3-linkage but not sialic acid in an α2,6-linkage(
      • Paulson J.C.
      • Weinstein J.
      • Dorland L.
      • Van Halbeek H.
      • Vliegenthart J.F.G.
      ), yielded a pattern identical with that obtained with 2 N acetic acid. Thus, 35% and 17% of the oligosaccharides on submaxillary CA VI contain 1 or 2 sulfate moieties, respectively, and sialic acid, when present, is exclusively in an α2,3-linkage.
      Figure thumbnail gr6
      Figure 6Anion exchange HPLC of Asn-linked oligosaccharides from submaxillary and parotid carbonic anhydrase VI. Asn-linked oligosaccharides from purified CA VI were released by treatment with PNGase F, purified, and labeled at their reducing termini with [3H]borohydride as outlined under “Experimental Procedures.” Labeled oligosaccharides from submaxillary (A) and parotid (B) CA VI were subjected to anion exchange HPLC on an AX-5 column. The elution positions of authentic oligosaccharides bearing no charged residues (N0), one (N1), two (N2), or three (N3) sialic acid residues; one (S1) or two (S2) sulfate residues; one sulfate and one sialic acid residue (SN), one sulfate and two sialic acid residues (S1N2), two sulfate and one sialic acid residue (S2N1), or a heterogeneous combination of sulfate and sialic acid residues (SxNy) producing four anionic charges are indicated.
      Anion exchange HPLC of the labeled oligosaccharides from parotid CA VI (Fig. 6B) yielded only 3 peaks corresponding to oligosaccharides with 0, 1, or 2 sialic acid moieties (N0, N1, and N2, respectively). Treatment with 2 N acetic acid or digestion with Newcastle disease virus neuraminidase converted all of the oligosaccharides to neutral species, indicating that these oligosaccharides were devoid of sulfate and that the sialic acid moieties were exclusively in an α2,3-linkage.
      The oligosaccharides from submaxillary and parotid CA VI were fractionated by affinity chromatography on WFA-agarose to determine the proportion of each oligosaccharide species which bear terminal β1,4-linked GalNAc. In the case of submaxillary CA VI, the neutral oligosaccharide fraction had the greatest proportion of structures bound by WFA-agarose (45%). The proportion of oligosaccharides bound by WFA-agarose declined as species with a greater number of anionic moieties were examined (Table 3). No change in the proportion of each oligosaccharide species bound by WFA-agarose was seen following digestions with neuraminidase indicating that the sialic acid present is not linked to GalNAc. In contrast, following digestion of those oligosaccharides identified as having one or more sulfate moieties on the basis of their elution time on anion exchange HPLC (Fig. 6A, species S1, SN, S2, S1N2, and S2N1) with recombinant GalNAc-4-sulfatase(
      • Litjens T.
      • Morris C.P.
      • Gibson G.J.
      • Beckmann K.R.
      • Hopwood J.J.
      ,
      • Anson D.S.
      • Muller V.
      • Bielicki J.
      • Harper G.S.
      • Hopwood J.J.
      ), ≥84% of each species was bound by WFA-agarose (Table 3). The presence of some S1N2 in the fraction designated N3 accounts for the increase in WFA-agarose binding of this fraction upon treatment with sulfatase. We have previously shown that terminal GalNAc in β1,4-linkage to GlcNAc can be released by digestion with jack bean β-hexosaminidase but not by digestion with diplococcal β-hexosaminidase(
      • Green E.D.
      • Van Halbeek H.
      • Boime I.
      • Baenziger J.U.
      ). Digestion of the desulfated oligosaccharides from submaxillary gland CA VI with jack bean β-hexosaminidase, but not diplococcal β-hexosaminidase, abolished binding to WFA-agarose confirming that the GalNAc exposed by digestion with GalNAc-4-sulfatase is in β1,4-linkage.
      The results described above indicate that 55% of the oligosaccharides released from submaxillary CA VI contain one or more branches terminating with the sequence SO4-4-GalNAcβ1,4GlcNAcβ. Among the oligosaccharides which are neutral or bear only sialic acid moieties, an additional 9% of the total have one or more branches terminating with GalNAcβ1,4GlcNAcβ. Therefore, at least 64% of the Asn-linked oligosaccharides obtained from submaxillary CA VI have either sulfated or nonsulfated termini containing β1,4-linked GalNAc. As was seen for the glycoprotein hormones(
      • Green E.D.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ), the addition of GalNAc to the Asn-linked oligosaccharides of submaxillary CA VI is a highly efficient process.
      The oligosaccharides from parotid CA VI were also fractionated into species that were bound and not bound to WFA-agarose. The bound fractions represented 52% of N0, 43% of N1, and 18% of N2 (Table 4). Digestion of the unbound species with Newcastle disease virus neuraminidase did not convert them to species which could bind WFA indicating that the sialic acid was not linked to GalNAc. Digestion with jack bean β-hexosaminidase but not with diplococcal β-hexosaminidase abolished WFA binding by the previously bound species. The properties of the WFA-bound species indicates that they contain at least one branch which terminates with the sequence GalNAcβ1,4GlcNAcβ. Thus, 40% of the Asn-linked oligosaccharides from parotid CA VI contains one or more branches which terminate with GalNAcβ1,4GlcNAcβ.
      A striking feature of the oligosaccharides from both submaxillary and parotid CA VI when examined by ion suppression amine adsorption-HPLC, which fractionates oligosaccharides on the basis of size as well as charge(
      • Mellis S.J.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      ), was their large size. Furthermore, the anionic oligosaccharides which contained either GalNAc-4-SO4 or terminal GalNAc from both submaxillary and parotid CA VI were not bound by ConA (not shown). This suggests that the Asn-linked oligosaccharides on submaxillary and parotid CA VI are more highly branched structures than the dibranched oligosaccharides typical of the glycoprotein hormones LH and TSH(
      • Green E.D.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ). Due to their heterogeneity and complexity, the detailed structures of these oligosaccharides have not yet been examined.

      DISCUSSION

      We originally described oligosaccharides terminating with the sequence SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα on the pituitary glycoprotein hormones LH and TSH and the uncombined glycoprotein hormone α subunit(
      • Green E.D.
      • Van Halbeek H.
      • Boime I.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      • Green E.D.
      ,
      • Stockell Hartree A.
      • Renwick A.G.C.
      ). Synthesis of these unique structures is accounted for by a GalNAc-transferase which displays peptide as well as oligosaccharide specificity (
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Smith P.L.
      • Baenziger J.U.
      ,
      • Mengeling B.J.
      • Manzella S.M.
      • Baenziger J.U.
      ) and by a GalNAc-4-sulfotransferase(
      • Skelton T.P.
      • Hooper L.V.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ,
      • Green E.D.
      • Baenziger J.U.
      • Boime I.
      ). Using highly specific GalNAc-transferase and GalNAc-4-sulfotransferase assays, we have detected both transferase activities in a number of tissues and cell lines(
      • Skelton T.P.
      • Kumar S.
      • Smith P.L.
      • Beranek M.C.
      • Baenziger J.U.
      ,
      • Dharmesh S.M.
      • Skelton T.P.
      • Baenziger J.U.
      ), suggesting that oligosaccharides terminating with GalNAc-4-SO4 may be present on glycoproteins unrelated to the glycoprotein hormones. Oligosaccharides bearing GalNAc linked β1,4 to an underlying GlcNAc have been subsequently described on glycoproteins from a number of different sources(
      • Nyame K.
      • Smith D.F.
      • Damian R.T.
      • Cummings R.D.
      ,
      • Sato T.
      • Furukawa K.
      • Greenwalt D.E.
      • Kobata A.
      ,
      • Chan A.L.
      • Morris H.R.
      • Panico M.
      • Etienne A.T.
      • Rogers M.E.
      • Gaffney P.
      • Creighton-Kempsford L.
      • Dell A.
      ,
      • Tanaka N.
      • Nakada H.
      • Itoh N.
      • Mizuno Y.
      • Takanishi M.
      • Kawasaki T.
      • Tate S.
      • Inagaki F.
      • Yamashina I.
      ,
      • Tomiya N.
      • Awaya J.
      • Kurono M.
      • Hanzawa H.
      • Shimada I.
      • Arata Y.
      • Yoshida T.
      • Takahashi N.
      ,
      • Yan S.B.
      • Chao Y.B.
      • Van Halbeek H.
      ,
      • Srivatsan J.
      • Smith D.F.
      • Cummings R.D.
      ,
      • Coddeville B.
      • Strecker G.
      • Wieruszeski J.M.
      • Vliegenthart J.F.G.
      • Van Halbeek H.
      • Peter-Katalinic J.
      • Egge H.
      • Spik G.
      ,
      • Bergwerff A.A.
      • Thomas-Oates J.E.
      • Van Oostrum J.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      ,
      • Kubelka V.
      • Altmann F.
      • Staudacher E.
      • Tretter V.
      • Marz L.
      • Hård K.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      ,
      • Nakata N.
      • Furukawa K.
      • Greenwalt D.E.
      • Sato T.
      • Kobata A.
      ,
      • Van Kuik J.A.
      • Sijbesma R.P.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      • Wood E.J.
      ); however, in addition to the glycoprotein hormones LH and TSH, oligosaccharides terminating with GalNAc-4-SO4 have been described only on POMC(
      • Skelton T.P.
      • Kumar S.
      • Smith P.L.
      • Beranek M.C.
      • Baenziger J.U.
      ,
      • Siciliano R.A.
      • Morris H.R.
      • McDowell R.A.
      • Azadi P.
      • Rogers M.E.
      • Bennett H.P.
      • Dell A.
      ,
      • Siciliano R.A.
      • Morris H.R.
      • Bennett H.P.J.
      • Dell A.
      ), recombinant TFPI(
      • Smith P.L.
      • Skelton T.P.
      • Fiete D.
      • Dharmesh S.M.
      • Beranek M.C.
      • MacPhail L.
      • Broze Jr., G.J.
      • Baenziger J.U.
      ), and Tamm-Horsfall glycoprotein purified from urine(
      • Hård K.
      • Van Zadelhoff G.
      • Moonen P.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      ). In contrast to POMC, TFPI, and the glycoprotein hormones, where one or more termini with GalNAc-4-SO4 are present on the majority of oligosaccharides isolated from these glycoproteins, less than 2% of the Asn-linked oligosaccharides present on Tamm-Horsfall glycoprotein terminate with GalNAc-4-SO4, making it a relatively minor component on this glycoprotein(
      • Hård K.
      • Van Zadelhoff G.
      • Moonen P.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      ).
      The present study demonstrates that Asn-linked oligosaccharides terminating with GalNAc-4-SO4 are the predominant structure on CA VI synthesized in submaxillary glands. High levels of GalNAc-transferase and GalNAc-4-sulfotransferase activities may be required in the submaxillary gland because the substrate, CA VI, is a major synthetic product. We have determined that Asn-linked oligosaccharides terminating with β1,4-linked GalNAc which is neither sulfated nor sialylated are the predominant structures present on CA VI synthesized in bovine parotid glands. CA VI is also a major product of the parotid gland which expresses high levels of GalNAc-transferase but no GalNAc-4-sulfotransferase. The absence of sulfate on parotid CA VI and the absence of GalNAc-4-sulfotransferase activity in parotid gland extracts indicate that this sulfotransferase accounts for sulfate addition to oligosaccharides on CA VI in the submaxillary gland. The results obtained with CA VI establish that there are endogenously synthesized proteins in tissues other than pituitary and unrelated to the glycoprotein hormones which bear oligosaccharides terminating with GalNAc-4-SO4. Furthermore, we expect that additional endogenous glycoproteins bearing this structure will be found in other tissues such as lacrimal gland and kidney which express relatively high levels of GalNAc-transferase and GalNAc-4-sulfotransferase.
      We have shown that digestion of submaxillary and parotid CA VI oligosaccharides with Newcastle disease virus neuraminidase results in the quantitative release of sialic acid from both forms. Since this neuraminidase is specific for sialic acid in α2,3 linkage, we conclude that all of the sialic acid found on the oligosaccharides from both tissue forms of CA VI is linked α2,3 to the underlying sugar. In addition, removal of sialic acid from both CA VI tissue forms does not expose any underlying GalNAc, indicating that sialic acid is linked to sugars other than GalNAc, predominantly Galβ1,4GlcNAcβ. This is consistent with our observation that α2,3-sialyltransferase, in contrast to α2,6-sialyltransferase(
      • Nemansky M.
      • Van den Eijnden D.H.
      ), does not transfer sialic acid to terminal β1,4-linked GalNAc in an in vitro assay. (
      S. M. Manzella and J. U. Baenziger, unpublished observation.
      )Since α2,6-sialyltransferase is able to transfer sialic acid to either β1,4-linked GalNAc or Gal (
      • Nemansky M.
      • Van den Eijnden D.H.
      ), the absence of detectable α2,6-linked sialic acid on either submaxillary or parotid CA VI oligosaccharides implies that α2,6-sialyltransferase is not expressed in cells synthesizing CA VI in either tissue. The absence of GalNAc-4-sulfotransferase and α2,6-sialyltransferase can thus account for the presence of terminal GalNAc, which is neither sulfated nor sialylated, on CA VI from bovine parotid glands.
      Expression of the glycoprotein hormone:GalNAc-transferase in bovine submaxillary and parotid glands in conjunction with the observation of β1,4-linked GalNAc in CA VI Asn-linked oligosaccharides suggests that the GalNAc-transferase in salivary glands is the same as the GalNAc-transferase we previously characterized as responsible for GalNAc addition to Asn-linked oligosaccharides on the glycoprotein hormones in pituitary(
      • Smith P.L.
      • Baenziger J.U.
      ). The requirements for peptide recognition by this GalNAc-transferase have been extensively characterized. In the case of the glycoprotein hormone α subunit, basic amino acids within the sequence Pro-Leu-Arg-Ser-Lys-Lys, which is amino-terminal to the first of two Asn-linked glycosylation sites, are found within an α-helix, thus forming a cluster of basic residues which is essential for recognition by the GalNAc-transferase(
      • Mengeling B.J.
      • Manzella S.M.
      • Baenziger J.U.
      ). The amino acid sequences of human and sheep CA VI have been established by cDNA (
      • Aldred P.
      • Fu P.
      • Barrett G.
      • Penschow J.D.
      • Wright R.D.
      • Coghlan J.P.
      • Fernley R.T.
      ) and protein (
      • Fernley R.T.
      • Wright R.D.
      • Coghlan J.P.
      ) sequencing, respectively. CA VI from both species contains good candidate sequences for recognition by the glycoprotein hormone:GalNAc-transferase. The sequence Pro-Lys-Arg-Lys-Lys is present 59 residues carboxyl-terminal to the nearest predicted Asn-linked glycosylation site (Asn239) in sheep CA VI, and the sequence Pro-Leu-Lys-His-Arg is present 12 residues carboxyl-terminal to a potential Asn-linked glycosylation site (Asn256) in human CA VI. The presence of good candidate sequences for recognition by the glycoprotein hormone:GalNAc-transferase along with the high levels of GalNAc-transferase activity found in the submaxillary and parotid glands strongly suggest that the glycoprotein hormone:GalNAc-transferase accounts for the presence of β1,4-linked GalNAc on CA VI. However, further studies will be required to determine whether these candidate sequences account for recognition of CA VI by the GalNAc-transferase.
      Submaxillary CA VI represents the first example of a naturally occurring glycoprotein not synthesized in the pituitary in which a major fraction of the Asn-linked oligosaccharides terminate with SO4-4-GalNAcβ1,4GlcNAcβ. The presence of the same terminal structure which is not sulfated on parotid CA VI suggests that critical biologic function(s) are associated with these oligosaccharides. In the case of LH, a receptor in hepatic endothelial cells recognizes oligosaccharides with terminal SO4-4-GalNAcβ1,4GlcNAcβ and rapidly removes LH from the circulation(
      • Fiete D.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      • Kumar S.
      • Brodbeck R.M.
      • Smith P.L.
      • Beranek M.C.
      ). As a result, LH has a shortened circulatory half-life which produces the pulsatile rise and fall of this hormone in the blood. Since CA VI is released into the saliva (
      • Feldstein J.B.
      • Silverman D.N.
      ,
      • Murakami H.
      • Sly W.S.
      ,
      • Fernley R.T.
      • Darling P.
      • Aldred P.
      • Wright R.D.
      • Coghlan J.P.
      ,
      • Parkkila S.
      • Kaunisto K.
      • Rajaniemi L.
      • Kumpulainen T.
      • Jokinen K.
      • Rajaniemi H.
      ,
      • Ogawa Y.
      • Chang C.-K.
      • Kuwakara H.
      • Hong S.-S.
      • Toyosawa S.
      • Yagi T.
      ) and not the bloodstream, it is not likely to encounter the hepatic receptor for GalNAc-4-SO4. The unique oligosaccharide structures on CA VI may have an antibacterial function as has been hypothesized for the heterogeneous oligosaccharides on mucins(
      • Tabak L.A.
      • Levine M.J.
      • Mandel I.D.
      • Ellison S.A.
      ). Alternatively, receptors similar to the GalNAc-4-SO4 receptor in hepatic endothelial cells (
      • Fiete D.
      • Srivastava V.
      • Hindsgaul O.
      • Baenziger J.U.
      ,
      • Baenziger J.U.
      • Kumar S.
      • Brodbeck R.M.
      • Smith P.L.
      • Beranek M.C.
      ) and the Gal/GalNAc-specific receptor in hepatocytes (
      • Ashwell G.
      • Harford J.
      ) may reside in different regions of the oral mucosa and selectively immobilize submaxillary and parotid CA VI, respectively. Immobilization in specific regions of the mouth could play a critical role in the localized regulation of oral pH.
      Even though the biologic function of the oligosaccharides on CA VI is not yet established, the different oligosaccharide structures found on submaxillary and parotid CA VI in conjunction with the pattern of transferase expression in these two tissues establish that: 1) glycoproteins bearing Asn-linked oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAcβ are synthesized outside the pituitary; 2) the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase are not always coordinately expressed in other tissues as they are in the pituitary; 3) the glycoprotein hormone:GalNAc-transferase, when expressed in the absence of GalNAc-4-sulfotransferase, can account for the synthesis of glycoproteins bearing oligosaccharides with terminal GalNAcβ1,4GlcNAcβ; and 4) glycoproteins not destined to enter the bloodstream may bear SO4-4-GalNAcβ1,4GlcNAcβ or GalNAcβ1,4GlcNAcβ. In addition, the selective transfer of different terminal sugars to oligosaccharides bearing GalNAcβ1,4GlcNAcβ may result in the synthesis of other unique oligosaccharides with yet other functions. The presence of glycoprotein hormone:GalNAc-transferase and/or GalNAc-4-sulfotransferase activity in a number of tissues and cell lines suggests that oligosaccharides related to those originally described on the glycoprotein hormones will be found on other glycoproteins of diverse functions.

      ACKNOWLEDGEMENTS

      We thank Dr. J. J. Hopwood for generously providing recombinant GalNAc-4-sulfatase.

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