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• W1968653306 abstract "Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2–5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15–30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm. Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2–5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15–30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm. adenosine 3′-phosphate 5′-phosphosulfate brefeldin A proteoglycans glycosaminoglycan chondroitin sulfate proteoglycan heparan sulfate proteoglycan Madin-Darby canine kidney Dulbecco's modified Eagle's medium fetal calf serum potorous tridactylis kidney Sulfation is a frequent Golgi modification found in glycoproteins (1Bauerle P.A. Huttner W.B. J. Cell Biol. 1987; 105: 2655-2664Crossref PubMed Scopus (195) Google Scholar), glycolipids (2Vos J.P. Lopes-Cardozo M. Gadella B.M. Biochim. Biophys. Acta. 1994; 1211: 125-149Crossref PubMed Scopus (193) Google Scholar), and proteoglycans (3Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1674) Google Scholar). Intracellular sulfate is provided by uptake via sulfate transporters in the plasma membrane (4Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar,5Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve-Daly M.P Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (622) Google Scholar). In the cytoplasm, sulfate is enzymatically activated to adenosine 3′-phosphate 5′-phosphosulfate (PAPS),1 which is translocated through specific transporter molecules (6Mandon E.C. Milla M.E. Kemper E. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10707-10711Crossref PubMed Scopus (62) Google Scholar, 7Ozeran J.D. Westley J. Schwartz N.B. Biochemistry. 1996; 35: 3685-3694Crossref PubMed Scopus (25) Google Scholar, 8Ozeran J.D. Westley J. Schwartz N.B. Biochemistry. 1996; 35: 3695-3703Crossref PubMed Scopus (32) Google Scholar) in the Golgi membrane and utilized as sulfate donor in the Golgi lumen by sulfotransferases (9Schwartz J.K. Capasso J.M. Hirschberg C.B. J. Biol. Chem. 1984; 259: 3554-3559Abstract Full Text PDF PubMed Google Scholar). Proteoglycans (PGs) are sulfated at various positions along their long linear glycosaminoglycan (GAG) chains that are attached to serines. Reduced (5Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve-Daly M.P Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (622) Google Scholar) or impaired (10Superti-Furga A. Hästbacka J. Wilcox W.R. Cohn D.H. van der Harten H.J. Rossi A. Blau N. Rimoin D.L. Steinmann B. Lander E.S. Gitzelmann R. Nat. Genet. 1996; 12: 100-102Crossref PubMed Scopus (187) Google Scholar) sulfate uptake across the plasma membrane is reported to be the primary defect in patients with mutations in the diastrophic dysplasia (DTD) gene. The resulting reduction in sulfation of PGs in cartilage matrix is the cause of the main clinical features connected with DTD (5Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve-Daly M.P Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (622) Google Scholar). Mutations in the same gene may give complete impairment of sulfate uptake, resulting in achondrogenisis type IB and perinatal death (10Superti-Furga A. Hästbacka J. Wilcox W.R. Cohn D.H. van der Harten H.J. Rossi A. Blau N. Rimoin D.L. Steinmann B. Lander E.S. Gitzelmann R. Nat. Genet. 1996; 12: 100-102Crossref PubMed Scopus (187) Google Scholar). No genetic disorder has been connected with later steps in the sulfation pathway, but the molecular basis is still unknown in the majority of osteochondrodysplasias. Localization of enzymatic activities to different regions of the Golgi apparatus has been facilitated by use of the fungal isoprenoid metabolite brefeldin A (BFA). In most cell types, secretory transport is inhibited by BFA because treatment with this drug induces retrograde transport of components of the cis-, medial, andtrans-Golgi cisternae but not of the trans-Golgi network to the endoplasmic reticulum (11Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 12Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar, 13Lippincott-Schwartz J. Donaldson J.G. Schweizer A. Berger E.G. Hauri H.P. Yuan L.C. Klausner R.D. Cell. 1990; 60: 821-836Abstract Full Text PDF PubMed Scopus (732) Google Scholar, 14Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (134) Google Scholar). In several cell types, the synthesis of HSPGs may be completed in the presence of BFA although with significantly reduced efficiency, whereas CSPG synthesis is not detected, which indicates that separate enzyme systems in early and late subcompartments of the Golgi complex are involved in HSPG and CSPG synthesis, respectively (15Spiro R.C. Freeze H.H. Sampath D. Garcia J.A. J. Cell Biol. 1991; 115: 1463-1473Crossref PubMed Scopus (64) Google Scholar, 16Sugumaran G. Katsman M. Silbert J.E. J. Biol. Chem. 1992; 267: 8802-8806Abstract Full Text PDF PubMed Google Scholar, 17Fransson L.-Å. Karlsson P. Schmidtchen A. Biochim. Biophys. Acta. 1992; 1137: 287-297Crossref PubMed Scopus (31) Google Scholar, 18Uhlin-Hansen L. Yanagishita M. J. Biol. Chem. 1993; 268: 17370-17376Abstract Full Text PDF PubMed Google Scholar, 19Calabro A. Hascall V.C. J. Biol. Chem. 1994; 269: 22764-22770Abstract Full Text PDF PubMed Google Scholar). Two kidney epithelial cell lines, MDCK and PtK, have Golgi stacks that are morphologically resistant to BFA treatment (14Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (134) Google Scholar, 20Ktistakis N.T. Roth M.G. Bloom G.S. J. Cell Biol. 1991; 113: 1009-1023Crossref PubMed Scopus (93) Google Scholar,21Hunziker W. Whitney J.A. Mellman I. Cell. 1991; 67: 617-627Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Despite this fact, apical protein secretion is reduced in MDCK cells already at low BFA concentrations, whereas basolateral secretion initially compensates for the reduction in apical secretion and is not inhibited until 20-fold higher BFA concentrations are applied (22Low S.H. Wong S.H. Tang B.L. Subramaniam V.N. Hong W. J. Biol. Chem. 1991; 266: 17729-17732Abstract Full Text PDF PubMed Google Scholar,23Low S.H. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1992; 118: 51-62Crossref PubMed Scopus (58) Google Scholar). Here we report a novel effect of BFA in MDCK cells. At low concentrations of BFA in which the Golgi apparatus is morphologically intact, the sulfation of proteins and PGs was dramatically reduced. This effect seemed to be caused by a reduction in the uptake of PAPS into the lumen of the Golgi apparatus. Lowering the cytoplasmic PAPS level by chlorate also resulted in reduced PAPS levels in the Golgi lumen but gave a different reduction profile in the incorporation of sulfate into CSPG, HSPG, and proteins. The reconstitution in vitro of glycosaminoglycan (GAG) chain synthesis by incubating MDCK Golgi fractions with UDP sugars, [35S]PAPS, Mg2+, and Mn2+ was impaired when the Golgi fractions were pretreated with BFA. This impairment was overcome by the addition of cytosol from pig brain, rat liver, or MDCK cells. Our results indicate that sulfation within the Golgi depends on a factor present both in the cytoplasm and on Golgi membranes. Bovine serum albumin,N-ethylmaleimide, ε-aminocaproic acid, phenylmethylsulfonyl fluoride, 2,4-dinitrophenylalanine, Dextran Blue, Triton X-100, and UDP sugars were all from Sigma. Chondroitin ABC lyase (EC 4.2.2.4.) was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Protein A-Sepharose 4B, DEAE-Sephacel, and Sephadex G-50 Fine and Superfine were obtained from Amersham Biosciences. [35S]Sulfate, [3H]glucosamine,14C-labeled molecular mass standards and Amplify were fromAmersham Biosciences. [35S]PAPS was obtained from Marco Maccarana (University of Uppsala, Uppsala, Sweden) or PerkinElmer Life Sciences. Brefeldin A was purchased from Epicentre Technologies (Madison, WI). NOVEX 4–20% Precast Tris glycine gels were purchased from NOVEX (Encinitas, CA). Sodium chlorate and other inorganic chemicals were purchased from Merck (Darmstadt, Germany) with the exception of guanidine hydrochloride, which was purchased from (Fluka, Buchs, Switzerland). All tissue culture plastics were purchased from Costar Europe (VC Badhoevedorp, Holland). DME medium, fetal calf serum (FCS), l-glutamine, and penicillin/streptomycin were purchased from B.I. Bio-Whittaker (Verviers, Belgium). Sulfate-free RPMI 1640 and DME media without Arg, Cys, Gln, Leu, Met, glucose, inositol, and phosphate and the necessary additives were purchased from Invitrogen. MDCKII cells were grown as described previously (24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar). The cells were established on polycarbonate filters (pore size 0.4 μm; diameter 24.5 mm, Costar Transwell) at a density of 106 cells/filter unless otherwise described. The cells were used for labeling 3–4 days later. An evaluation of filter-grown epithelial monolayers and [35S]sulfate labeling in sulfate-free medium was carried out as described previously (25Svennevig K. Prydz K. Kolset S.O. Biochem. J. 1995; 311: 881-888Crossref PubMed Scopus (41) Google Scholar). Labeling with [3H]glucosamine was carried out in DME medium without Arg, Cys, Gln, Leu, Met, glucose, inositol, and phosphate supplemented with all additives with the exception of glucose. After metabolic labeling with [35S]sulfate or [3H]glucosamine, the medium fractions were harvested, eventual free cells were removed by centrifugation, and an equal volume of 8 m guanidine, 4% Triton X-100, 0.1 msodium acetate buffer, pH 6.0, was added. The cell fraction was washed twice (5 min each) with ice-cold phosphate-buffered saline and solubilized in 4 m guanidine, 2% Triton X-100, 0.05m sodium acetate buffer, pH 6.0. To measure the level of [35S]sulfate incorporated into macromolecules, 1 ml of each fraction was applied to a 4-ml column of Sephadex G-50 Fine in 0.05 m Tris/HCl, pH 8.0, 0.15 m NaCl. The first 1 ml of elute after application was discarded, the next 1.5 ml was collected, and an aliquot of this elute was counted for radioactivity in the scintillation counter. Free [35S] remained associated with the column, and the exchange of guanidine with Tris buffer made further analysis possible. 35S-Labeled macromolecules from cell and medium fractions obtained by Sephadex G-50 Fine chromatography were subjected to preparative ion-exchange chromatography. The samples were loaded onto columns (4-ml wet gel volume) in 0.05 m Tris/HCl, 0.15 m NaCl, pH 8.0, and washed with the same buffer. A gradient extending from 0.15 to 1.5 m NaCl in 0.05 m Tris/HCl then was applied. Fractions of 2 ml were collected from the start of the chromatography. Aliquots of each fraction were counted for radioactivity in a scintillation counter (1900 TR, Packard, Downers Grove, IL) after the addition of Ultima Gold AB scintillation fluid (Packard). Fractions containing peaks of 35S-labeled material were pooled and dialyzed against water at 4 °C with a mixture of protease inhibitors containing 10 mm EDTA, 1 mm ε-aminocaproic acid, 1 mm N-ethylmaleimide, and 1 mm phenylmethylsulfonyl fluoride. Dialyzed samples were frozen until further analysis. Samples with 35S-labeled or3H-labeled PGs and proteins were boiled in sample buffer with 1% SDS and applied to precast 4–20% gradient NuPAGE gels from Novex. Standards used were 14C-labeled rainbow standards from Amersham. After electrophoresis, the gels were fixed, treated with Amplify, dried, and subjected to autoradiography using Fuji Medical x-ray film (Tokyo, Japan). MDCKII cells were labeled with [35S]sulfate (0.1 mCi/ml) or [3H]glucosamine (0.2 mCi/ml) for 20 h after which the apical and basolateral media and the cell fractions were harvested. The cell fraction was solubilized directly into 0.05 mTris/HCl, pH 7.5, with 1% Nonidet P-40, 2 mm EDTA, 150 mm NaCl, 35 μg/ml phenylmethylsulfonyl fluoride. The three fractions obtained were incubated overnight at 4 °C with a rabbit antiserum against mouse Perlecan (Dr. J. R. Hassell, Shriners Hospital, Tampa, FL). The fractions had been supplemented with 5 mm MgSO4 or 5 mm glucose to reduce unspecific binding of free [35S]sulfate and [3H]glucosamine, respectively. The samples were subsequently incubated with protein A-Sepharose prewashed with phosphate-buffered saline containing 1% bovine serum albumin. The samples were washed and finally run on 4–20% Novex gradient gels. After electrophoresis, bands were visualized by autoradiography. Samples from media and cell fractions of ∼10,000 cpm were incubated with 0.01 units of enzyme as described previously (25Svennevig K. Prydz K. Kolset S.O. Biochem. J. 1995; 311: 881-888Crossref PubMed Scopus (41) Google Scholar). The degraded material represented chondroitin/dermatan sulfate, and the samples were compared with untreated samples by SDS-PAGE by gel filtration chromatography on Superose-6 columns (Amersham Biosciences). The amount of HSPG was determined by degradation with nitrous acid at pH 1.5, as described by Shively and Conrad (26Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (666) Google Scholar). NaOH treatment releases GAG, CS and HS chains from their protein cores by β-elimination. Samples (100–800 μl) of medium and cell fractions (in guanidine) were added to one-tenth of the sample volume of 5 m NaOH and incubated overnight at room temperature. The incubation was terminated by the addition of 5 m HCl to pH 7.0–8.0. The lengths of the free GAG chains were analyzed by Sepharose Cl-6B column chromatography. The elution volumes and K av coefficients were determined relatively to the elution of the V o marker Dextran Blue and the Vt marker 2,4-dinitrofenylalanin or K2CrO4. Golgi-enriched subcellular fractions from control and BFA-treated MDCKII cells were prepared as described previously (14Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (134) Google Scholar, 24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar). Cells were grown to confluency in 75-cm2 plastic flasks (Costar) in DME medium (24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar). In each experiment, two flasks were treated for 60 min with 1 μg/ml BFA in minimum Eagle's medium (Invitrogen) with 10 mm HEPES, pH 7.4, whereas two flasks were incubated in minimum Eagle's medium with 10 mm HEPES, pH 7.4, alone. After homogenization, a post-nuclear supernatant was made and 840 μl of this supernatant was mixed with 660 μl of 2 m sucrose, 10 mm CsCl, 1 mm HEPES, and this mixture was applied above a 6-ml gradient in SW 41 tubes as described previously (24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar). Two more layers were applied; first 3 ml of 0.9 m sucrose, 1 mmHEPES, and finally 2 ml of homogenization buffer (0.3 msucrose, 3 mm imidazole, pH 7.4). Golgi components could be recovered from the interphase between the two latter layers after centrifugation (33,000 rpm, 4.5 h in a Beckman SW 41 rotor). Incubation of Golgi fractions with [35S]PAPS was performed according to the translocation assay described by Brändli et al. (27Brändli A.W. Hansson G.C. Roudriguez-Boulan E. Simons K. J. Biol. Chem. 1988; 263: 16283-16290Abstract Full Text PDF PubMed Google Scholar). Variable amounts of Golgi protein were incubated for 30 min at 37 °C with 25 × 105 dpm [35S]PAPS in incubation buffer: 0.25 m sucrose, 150 mm KCl, 1 mm MgCl2, 10 mm Tris/HCl, pH 7.5, in a total volume of 1 ml. The reaction was stopped by the addition of 2 ml of ice-cold incubation buffer, and Golgi vesicles were sedimented by centrifugation (40,000 rpm/60 min) TFT 65.13 rotor (Kontron). The supernatant was removed, and the pellet was carefully resuspended and repelleted by centrifugation. The pellet was subsequently dissolved in 50 mm Tris/HCl, pH 7.5, 1% Nonidet P-40, 2 mm EDTA, 150 mm NaCl, 35 μg/ml phenylmethylsulfonyl fluoride, and the radioactivity incorporated in the pellet was determined in a 1900 TR scintillation counter (Packard) after the addition of Ultima Gold AB scintillation fluid (Packard). Protein was measured with the Biuret method or with the Lowry method as modified by Bensadoun and Weinstein (28Bensaduon A. Weinstein D. Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2735) Google Scholar). Golgi fractions were isolated as described previously (14Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (134) Google Scholar,24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar) but in larger scale. Cells were grown to confluency in 500-cm2 square tissue culture dishes (Corning/Costar) in DME medium with 5% FCS (24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar). In each experiment, one plate was treated overnight (20 h) with 2 μg/ml BFA in DME medium with FCS, whereas one plate was incubated in DME medium without BFA. After homogenization, a post-nuclear supernatant was made and 8.4 ml of this supernatant was mixed with 6.6 ml of 2 m sucrose, 10 mm CsCl, 1 mm HEPES, and this mixture was applied above a 10-ml gradient in SW 28 tubes (as described in previous paragraph). Two more layers were applied; first 5 ml of 0.9 m sucrose, 1 mm HEPES, and finally 5 ml of homogenization buffer (0.3m sucrose, 3 mm imidazole, pH 7.4). Golgi components could be recovered from the interphase between the two latter layers after centrifugation (28,000 rpm, 4.5 h in a Beckman SW 28 rotor). The Golgi fraction was recovered as described previously (14Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (134) Google Scholar, 24Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (87) Google Scholar) and incubated with UDP-glucuronic acid, UDP-N-acetylglucosamine, N-acetylgalactosamine, MgCl2, MnCl2, and [35S]PAPS for 2 h at 37 °C. The concentrations are indicated in each experiment. 35S-Labeled macromolecules were isolated by Sephadex G-50 Fine as described above and determined by scintillation counting and, in some cases, analyzed by SDS-PAGE. Pig brain cytosol was isolated as described previously (29Garred Ø. Rodal S.K. vanDeurs B. Sandvig K. Traffic. 2001; 2: 26-36Crossref PubMed Scopus (31) Google Scholar). Sulfation involves the transfer of sulfate from PAPS to substrates for sulfotransferases located both in the lumen of the Golgi apparatus and in the cytosol. Glycolipids, glycoproteins, and PGs in transit to the cell surface are substrates for sulfotransferases localized to different regions of the Golgi apparatus. Sulfation within the Golgi requires efficient uptake of PAPS from its site of synthesis in the cytosol into the Golgi lumen. This uptake is mediated via PAPS transporters within the Golgi membrane (6Mandon E.C. Milla M.E. Kemper E. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10707-10711Crossref PubMed Scopus (62) Google Scholar, 22Low S.H. Wong S.H. Tang B.L. Subramaniam V.N. Hong W. J. Biol. Chem. 1991; 266: 17729-17732Abstract Full Text PDF PubMed Google Scholar, 23Low S.H. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1992; 118: 51-62Crossref PubMed Scopus (58) Google Scholar). In our study, we have inhibited the incorporation of sulfate into proteins and PGs in two ways. One approach has been to inhibit the synthesis of PAPS in the cytoplasm by the addition of chlorate (30Baeuerle P.A. Huttner W.B. Biochem. Biophys. Res. Comm. 1986; 141: 870-877Crossref PubMed Scopus (292) Google Scholar, 31Kreuger J. Prydz K. Petterson R.F. Lindahl U. Salmivirta M. Glycobiology. 1999; 9: 723-729Crossref PubMed Scopus (77) Google Scholar, 32Safayian F. Kolset S.O. Prydz K. Gottfridsson E. Lindahl U. Salmivirta M. J. Biol. Chem. 1999; 274: 36267-36273Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Low concentrations of chlorate (2–5 mm) reduced the sulfation of proteins and CSPGs, whereas the sulfation of HSPGs was unaffected at these chlorate concentrations but could be inhibited at higher concentrations of chlorate (15–30 mm). Fig.1 shows the pattern of incorporation of [35S]sulfate into macromolecules in the apical medium, the basolateral medium, and the cell fraction after the treatment of MDCK cells with different concentrations of chlorate during the labeling period. PGs are seen as broad bands in the high molecular weight region of the gel, whereas sulfated proteins are seen as more distinct bands with lower molecular weights. Clearly, protein sulfation is reduced by 2 mm chlorate and is essentially undetectable with 5 mm chlorate in all three fractions. Sulfation of PGs is still efficient at 30 mm chlorate although somewhat reduced. To determine whether sulfation of CSPGs and HSPGs was affected differently by chlorate treatment, the PGs were separated from proteins by DEAE ion-exchange chromatography and subjected to chondroitinase ABC or HNO2 treatment to degrade CSPGs or HSPGs, respectively. Fig. 2 shows the result from the apical medium. With increasing concentrations of chlorate, the relative fraction of PGs (i.e. HSPG) is increasing, and already with 5 mm chlorate, almost all of the PG is HSPG and very little is CSPG. At all of the higher concentrations of chlorate, only HSPGs were detected (data not shown). The results for the cell fractions were similar to those for the apical medium, whereas the basolateral medium contained essentially only HSPG (data not shown) (33Kolset S.O. Vuong T.T. Prydz K. J. Cell Sci. 1999; 112: 1797-1801Crossref PubMed Google Scholar). These results indicate that in MDCK cells, HSPG sulfation, which generally is completed before the trans-Golgi network, may operate at a lower cellular PAPS concentration than sulfation events (protein and CS) that take place in the trans-Golgi network (15Spiro R.C. Freeze H.H. Sampath D. Garcia J.A. J. Cell Biol. 1991; 115: 1463-1473Crossref PubMed Scopus (64) Google Scholar, 16Sugumaran G. Katsman M. Silbert J.E. J. Biol. Chem. 1992; 267: 8802-8806Abstract Full Text PDF PubMed Google Scholar, 17Fransson L.-Å. Karlsson P. Schmidtchen A. Biochim. Biophys. Acta. 1992; 1137: 287-297Crossref PubMed Scopus (31) Google Scholar, 18Uhlin-Hansen L. Yanagishita M. J. Biol. Chem. 1993; 268: 17370-17376Abstract Full Text PDF PubMed Google Scholar, 19Calabro A. Hascall V.C. J. Biol. Chem. 1994; 269: 22764-22770Abstract Full Text PDF PubMed Google Scholar). Although PAPS K m values for MDCK cell sulfotransferases have not been determined, such K mvalues determined for sulfotransferases involved in HS synthesis in other cell types are considerably lower (0.2–2.4 μm) (34Kobayashi M. Habuchi H. Habuchi O. Saito M. Kimata K. J. Biol. Chem. 1996; 271: 7645-7653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 35Habuchi H. Habuchi O. Kimata K. J. Biol. Chem. 1995; 270: 4172-4179Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) than those involved in CS synthesis (40 μm for 6-sulfation) (36Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 37Sugumaran G. Katsman M. Drake R.R. J. Biol. Chem. 1995; 270: 22483-22487Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Chlorate acts as a specific inhibitor of sulfation, because the incorporation of [3H]glucosamine into macromolecules was unaffected in the presence of 5 and 20 mm chlorate (Fig. 3).Figure 2Effect of chlorate on the synthesis of chondroitin sulfate and heparan sulfate. Filter-grown MDCKII cells were incubated for 22 h with 0.1 mCi/ml [35S]sulfate without chlorate or in the presence of 2 or 5 mm chlorate in sulfate-free medium. At the end of the incubation period, the media were harvested and macromolecules were isolated by Sephadex G-50 Fine gel filtration before PGs were separated from proteins by DEAE ion-exchange chromatography. Pooled and dialyzed PG fractions were analyzed by Superose 6 chromatography. Untreated samples of media and cells and samples treated with nitrous acid or chondroitinase ABC were eluted in 0.05 m Tris/HCl, pH 8.0, 0.15 m NaCl, 0.5% Triton X-100. The figure shows the result for the apical medium. One experiment representative of three is shown. ●, untreated sample; ○, chondroitinase ABC-treated sample; ×, nitrous acid-treated sample.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Incorporation of [3H]glucosamine into macromolecules is unaffected by chlorate. MDCKII cells were labeled for 22 h in glucose-free medium with 2% FCS and 200 μCi of [3H]glucosamine added basolaterally to each filter. Apical (1 ml) and basolateral (2 ml) media were harvested, and the cell layer was solubilized. Labeled macromolecules were isolated on Sephadex G-50 Fine columns and analyzed by SDS-PAGE and autoradiography. One of two similar experiments is shown. The migration distances of the protein standards are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BFA has been shown to inhibit apical transport of glycoproteins in MDCK cells (22Low S.H. Wong S.H. Tang B.L. Subramaniam V.N. Hong W. J. Biol. Chem. 1991; 266: 17729-17732Abstract Full Text PDF PubMed Google Scholar, 23Low S.H. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1992; 118: 51-62Crossref PubMed Scopus (58) Google Scholar) without reducing the basolateral counterpart. Filter-grown MDCK cells were labeled with [35S]sulfate in the absence or presence of various concentrations of BFA (0.5, 1.0, 2.0, 3.0, and 5.0 μg/ml). After 20 h, macromolecules in the media and the cell fraction were separated from free [35S]sulfate. Radioactively labeled molecules in the eluted fractions were analyzed by SDS-PAGE. BFA treatment resulted in a decrease in sulfate-labeled macromolecules in both the apical medium, the basolateral medium, and the cell fraction (Fig. 4). The reduction, however, was first observed for the apical medium. This effect could be a combination of reduced sulfation and reduced apical secretion. We next wanted to investigate whether the effect of BFA was limited to the sulfation steps in GAG synthesis. To answer this question, we studied the incorporation of [3H]glucosamine and [35S" @default.
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