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• W2068208889 abstract "Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1+/− and NDST1−/− MCs is higher (≈30%) than in control MCs where ≈95% of the 35S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results. Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1+/− and NDST1−/− MCs is higher (≈30%) than in control MCs where ≈95% of the 35S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results. Mast cells (MCs) 4The abbreviations used are: MCmast cellCPA3carboxypeptidase A3Eembryonic dayEBembryoid bodyHSheparan sulfateMCPTmouse mast cell proteaseNDSTN-deacetylase/N-sulfotransferasermrecombinant mouseSCFstem cell factorRPIPreversed-phase ion pair. can be divided into mucosal type and connective tissue type MCs. The two types of MC differ in their content of several components including MC proteases (1Gurish M.F. Austen K.F. J. Exp. Med. 2001; 194: F1-F5Crossref PubMed Google Scholar). In rodents, the two types of MC can also be distinguished based on glycosaminoglycan content; whereas mucosal MCs synthesize the proteoglycan serglycin with chondroitin sulfate chains, serglycin in connective tissue type MCs contains heparin (2Kolset S.O. Prydz K. Pejler G. Biochem. J. 2004; 379: 217-227Crossref PubMed Scopus (136) Google Scholar). Also, during connective tissue type MC differentiation, glycosaminoglycan content differs between immature cells containing chondroitin sulfate and more highly differentiated cells that synthesize heparin (2Kolset S.O. Prydz K. Pejler G. Biochem. J. 2004; 379: 217-227Crossref PubMed Scopus (136) Google Scholar). mast cell carboxypeptidase A3 embryonic day embryoid body heparan sulfate mouse mast cell protease N-deacetylase/N-sulfotransferase recombinant mouse stem cell factor reversed-phase ion pair. Heparin is a highly sulfated variant of heparan sulfate (HS). Whereas heparin is found exclusively in the MC granules, HS is a ubiquitous component of cell surfaces and is also present in the extracellular matrix, predominantly in basement membranes. HS/heparin biosynthesis is a complex process with many different Golgi enzymes involved (3Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). The HS-polymerases EXT1 and EXT2 synthesize a polysaccharide backbone consisting of repeating units of glucuronic acid and N-acetylglucosamine. The first modifying event is the N-deacetylase/N-sulfotransferase (NDST) reaction, where N-acetyl groups of selected N-acetylglucosamine residues are removed and replaced by sulfate groups (4Grobe K. Ledin J. Ringvall M. Holmborn K. Forsberg E. Esko J.D. Kjellén L. Biochim. Biophys. Acta. 2002; 1573: 209-215Crossref PubMed Scopus (127) Google Scholar). After N-sulfation, the C5-epimerase converts glucuronic acid residues to iduronic acid. Sulfation at the 2-O position of iduronic acid residues and some glucuronic acid is then carried out by a 2-O-sulfotransferase, followed by glucosamine 6-O-sulfation and, more rarely, 3-O-sulfation. Little is known about the organization of the biosynthesis enzymes in the Golgi stacks. It has been suggested that the enzymes together with other yet unidentified components form GAGosomes, molecular machines responsible for elongation as well as modification of the glycosaminoglycan chains (5Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1217) Google Scholar). Different compositions of the GAGosome may result in different modification patterns of the HS chain. In support of the GAGosome concept, several interactions between pairs of HS/heparin biosynthesis enzymes have been demonstrated. For example, the polymerases EXT1 and EXT2 are known to form a functional complex (6McCormick C. Duncan G. Goutsos K.T. Tufaro F. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 668-673Crossref PubMed Scopus (362) Google Scholar, 7Senay C. Lind T. Muguruma K. Tone Y. Kitagawa H. Sugahara K. Lidholt K. Lindahl U. Kusche-Gullberg M. EMBO Rep. 2000; 1: 282-286Crossref PubMed Scopus (139) Google Scholar). Interactions have also been demonstrated between the C5-epimerase and the 2-O-sulfotransferase (8Pinhal M.A. Smith B. Olson S. Aikawa J. Kimata K. Esko J.D. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12984-12989Crossref PubMed Scopus (134) Google Scholar) and between the xylosyltransferase and galactosyltransferase-I (9Schwartz N.B. Rodén L. Dorfman A. Biochem. Biophys. Res. Commun. 1974; 56: 717-724Crossref PubMed Scopus (32) Google Scholar). In addition, we recently demonstrated an interaction between NDST1 and EXT2 that greatly influenced the structure of the polysaccharide formed (10Presto J. Thuveson M. Carlsson P. Busse M. Wilén M. Eriksson I. Kusche-Gullberg M. Kjellén L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4751-4756Crossref PubMed Scopus (133) Google Scholar). During recent years it has become evident that HS has a key role in embryonic development (11Lin X. Development. 2004; 131: 6009-6021Crossref PubMed Scopus (520) Google Scholar). As first demonstrated for FGFs, the proteoglycans act as co-receptors for signaling molecules (12Pellegrini L. Curr. Opin. Struct. Biol. 2001; 11: 629-634Crossref PubMed Scopus (235) Google Scholar). In addition, HS has been shown to create and maintain gradients of morphogens and cytokines (13Lander A.D. Nie Q. Wan F.Y. Dev. Cell. 2002; 2: 785-796Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Also, extracellular matrix proteins, proteases, protease inhibitors, lipases, lipoproteins, and microbial proteins are known to show affinity for HS (5Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1217) Google Scholar). Knock-out mice have been particularly useful to study the role of HS in development. Targeted disruption of the EXT1 and EXT2 genes, respectively, leads to a complete lack of HS and an early embryonic lethality (14Lin X. Wei G. Shi Z. Dryer L. Esko J.D. Wells D.E. Matzuk M.M. Dev. Biol. 2000; 224: 299-311Crossref PubMed Scopus (341) Google Scholar, 15Stickens D. Zak B.M. Rougier N. Esko J.D. Werb Z. Development. 2005; 132: 5055-5068Crossref PubMed Scopus (206) Google Scholar). Four different NDSTs have been recognized (4Grobe K. Ledin J. Ringvall M. Holmborn K. Forsberg E. Esko J.D. Kjellén L. Biochim. Biophys. Acta. 2002; 1573: 209-215Crossref PubMed Scopus (127) Google Scholar). Both NDST1 and NDST2 have broad expression patterns and are found in most cell types and tissues during embryonic development and adult life, whereas NDST3 and NDST4 have a much more restricted expression pattern (16Aikawa J. Grobe K. Tsujimoto M. Esko J.D. J. Biol. Chem. 2001; 276: 5876-5882Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Mouse strains deficient in NDST1 (17Ringvall M. Ledin J. Holmborn K. van Kuppevelt T. Ellin F. Eriksson I. Olofsson A.M. Kjellen L. Forsberg E. J. Biol. Chem. 2000; 275: 25926-25930Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 18Fan G. Xiao L. Cheng L. Wang X. Sun B. Hu G. FEBS Lett. 2000; 467: 7-11Crossref PubMed Scopus (141) Google Scholar, 19Grobe K. Inatani M. Pallerla S.R. Castagnola J. Yamaguchi Y. Esko J.D. Development. 2005; 132: 3777-3786Crossref PubMed Scopus (158) Google Scholar) have been characterized extensively (17Ringvall M. Ledin J. Holmborn K. van Kuppevelt T. Ellin F. Eriksson I. Olofsson A.M. Kjellen L. Forsberg E. J. Biol. Chem. 2000; 275: 25926-25930Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 18Fan G. Xiao L. Cheng L. Wang X. Sun B. Hu G. FEBS Lett. 2000; 467: 7-11Crossref PubMed Scopus (141) Google Scholar, 19Grobe K. Inatani M. Pallerla S.R. Castagnola J. Yamaguchi Y. Esko J.D. Development. 2005; 132: 3777-3786Crossref PubMed Scopus (158) Google Scholar, 20Ledin J. Staatz W. Li J.P. Götte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 21Ledin J. Ringvall M. Thuveson M. Eriksson I. Wilén M. Kusche-Gullberg M. Forsberg E. Kjellén L. J. Biol. Chem. 2006; 281: 35727-35734Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Wang L. Fuster M. Sriramarao P. Esko J.D. Nat. Immunol. 2005; 6: 902-910Crossref PubMed Scopus (376) Google Scholar, 23Pan Y. Woodbury A. Esko J.D. Grobe K. Zhang X. Development. 2006; 133: 4933-4944Crossref PubMed Scopus (86) Google Scholar, 24Pallerla S.R. Pan Y. Zhang X. Esko J.D. Grobe K. Dev. Dyn. 2007; 236: 556-563Crossref PubMed Scopus (49) Google Scholar, 25Fuster M.M. Wang L. Castagnola J. Sikora L. Reddi K. Lee P.H. Radek K.A. Schuksz M. Bishop J.R. Gallo R.L. Sriramarao P. Esko J.D. J. Cell Biol. 2007; 177: 539-549Crossref PubMed Scopus (99) Google Scholar, 26MacArthur J.M. Bishop J.R. Stanford K.I. Wang L. Bensadoun A. Witztum J.L. Esko J.D. J. Clin. Invest. 2007; 117: 153-164Crossref PubMed Scopus (166) Google Scholar, 27Hu Z. Yu M. Hu G. Bone. 2007; 40: 1462-1474Crossref PubMed Scopus (18) Google Scholar, 28Abramsson A. Kurup S. Busse M. Yamada S. Lindblom P. Schallmeiner E. Stenzel D. Sauvaget D. Ledin J. Ringvall M. Landegren U. Kjellén L. Bondjers G. Li J.P. Lindahl U. Spillmann D. Betsholtz C. Gerhardt H. Genes Dev. 2007; 21: 316-331Crossref PubMed Scopus (138) Google Scholar, 29Pan Y. Carbe C. Powers A. Zhang E.E. Esko J.D. Grobe K. Feng G.S. Zhang X. Development. 2008; 135: 301-310Crossref PubMed Scopus (81) Google Scholar, 30Hu Z. Wang C. Xiao Y. Sheng N. Chen Y. Xu Y. Zhang L. Mo W. Jing N. Hu G. J. Cell Sci. 2009; 122: 1145-1154Crossref PubMed Scopus (35) Google Scholar, 31Ringvall M. Kjellén L. Prog. Mol. Biol. Transl. Sci. 2010; 93: 35-58Crossref PubMed Scopus (21) Google Scholar). Complete lack of NDST1 results in perinatal lethality, forebrain defects, skeletal malformation, and lung hypoplasia. Vascular development, endothelial cell function, lipid metabolism, lacrimal gland induction, lens development, and neural tube fusion have also been shown to be impaired in NDST1-deficient animals. In contrast, NDST2-deficient mice are healthy and fertile, but their connective tissue type MCs lack sulfated heparin and contain reduced levels of histamine and the MC-specific proteases, (32Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellén L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (403) Google Scholar, 33Humphries D.E. Wong G.W. Friend D.S. Gurish M.F. Qiu W.T. Huang C. Sharpe A.H. Stevens R.L. Nature. 1999; 400: 769-772Crossref PubMed Scopus (358) Google Scholar). Also, mice with a targeted deletion of NDST3 develop normally with only subtle symptoms, such as lowered cholesterol and HDL levels (34Pallerla S.R. Lawrence R. Lewejohann L. Pan Y. Fischer T. Schlomann U. Zhang X. Esko J.D. Grobe K. J. Biol. Chem. 2008; 283: 16885-16894Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). No knock-out strain carrying a targeted deletion of NDST4 has yet been described. Crosses between NDST2−/− mice and NDST1+/− and analyses of their offspring demonstrated that lack of both isoforms results in early embryonic lethality (35Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To be able to determine the cause of the early embryonic lethality, we recently established embryonic stem (ES) cell lines deficient for both NDST1 and NDST2 and showed that the HS produced lacks N-sulfation but contains low levels of 6-O-sulfate groups (35Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the present paper, we have studied the role of NDST1 and NDST2 in MC development in vitro. Although no MCs were formed when ES cells deficient in both NDST1 and NDST2 were differentiated in vitro, deficiency in either NDST1 or NDST2 was compatible with MC differentiation. In vitro differentiated NDST2−/− MCs had an altered morphology compared with control cells and lacked or showed reduced expression of MC proteases. These cells synthesized HS/heparin with a lower sulfation degree than polysaccharide isolated from control cells. In contrast, in vitro differentiated NDST1−/− as well as NDST1+/− MCs contained increased levels of connective tissue type MC proteases compared with control cells and synthesized heparin with a higher degree of sulfation. For the generation of MCs from mouse embryos the following mouse strains were used: C57BL/6, NDST1+/− (N10 on C57BL/6) (17Ringvall M. Ledin J. Holmborn K. van Kuppevelt T. Ellin F. Eriksson I. Olofsson A.M. Kjellen L. Forsberg E. J. Biol. Chem. 2000; 275: 25926-25930Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and NDST1+/−2−/− (N10 on C57BL/6) (32Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellén L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (403) Google Scholar). NDST1+/−2−/− mice were obtained by crossing NDST1+/− with NDST2−/− mice. All animal experiments were conducted with the approval of the local animal ethical committee in Uppsala. Embryo-derived MCs were obtained by culturing of day 10.5 to day 12.5 embryos in defined medium in a humidified cell culture chamber in 37 °C and 5% CO2 essentially as described by Vial et al. (36Vial D. Oliver C. Jamur M.C. Pastor M.V. da Silva Trindade E. Berenstein E. Zhang J. Siraganian R.P. J. Immunol. 2003; 171: 6178-6186Crossref PubMed Scopus (12) Google Scholar). The embryos were collected and transferred into a 24-well plate containing 0.5 ml of Complete medium (DMEM supplemented with 10% heat-inactivated FBS, 4 mm l-glutamine, 0.5 × 10−6 m β-mercaptoethanol, 10% NCTC 109 medium (Invitrogen), 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 50 μg/ml G418, 10 ng/ml recombinant mouse (rm) IL-3 and 25 ng/ml rmSCF). Embryo tissues were left to adhere to the plastic for 2 days and then trypsinized to disrupt embryonic structures and to get a dispersed cell suspension. Trypsinization was stopped by addition of 1.5 ml (10× the volume of trypsin) of complete medium. The dispersed cells quickly adhered to the tissue culture dish, and after ∼4 days to 1 week nonadherent cells appearing in the cultures were transferred into 6-well plates and expanded for 2–4 weeks. Aliquots of cells were removed from the developing MC culture corresponding to each embryo. Cells were put on cytospin glasses and stained with May-Grünwald/Giemsa for morphological examination of MC differentiation. In addition, DNA was purified and analyzed by PCR, as described previously (35Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), to define the genotype of the cultures. NDST1−/−2−/− ES cells were cultivated as described previously (35Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). NDST1+/−, NDST2+/−, and WT (R1) (37Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8424-8428Crossref PubMed Scopus (1954) Google Scholar) ES cells were used as controls. Before differentiation, ES cells were cultured in DMEM with GlutaMAX-1, sodium pyruvate, 4,500 mg/liter glucose, and pyridoxine (Invitrogen) supplemented with 1× nonessential amino acids (Invitrogen), 20% FCS (ES cell qualified; Invitrogen), 10−4 m β-mercaptoethanol (Sigma), and 1,000 units/ml leukemia inhibitory factor (mouse recombinant, Chemicon). To induce MC development, a slight modification of the protocol described previously by Tsai et al. was used (38Tsai M. Wedemeyer J. Ganiatsas S. Tam S.Y. Zon L.I. Galli S.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9186-9190Crossref PubMed Scopus (78) Google Scholar). In different experiments, 2,000 or 20,000 ES cells were plated in bacterial dishes in 100-μl droplets of Iscove's modified Dulbecco's medium (Sigma) containing 15% FCS, 2 mm l-glutamine (Sigma), 5 ng/ml rmIL-11 (R&D Systems), 50 ng/ml rmSCF (Peprotech), 50 μg/ml G418 (Invitrogen), and 450 μm monothioglycerol (Sigma) to enable formation of embryoid bodies (EBs). The plating of cells on nonadherent bacterial Petri dishes is referred to as day 0. Two to 3 days after the initiation of EB formation, more medium was added to achieve a floating EB culture. Six days after initiation, the EB cultures were boosted with 1 volume of Iscove's modified Dulbecco's medium containing 15% FCS, 2 mm l-glutamine, 30 ng/ml rmIL-6 (Peprotech), 30 ng/ml rmIL-3 (Peprotech), 50 ng/ml rmSCF (Peprotech), 50 μg/ml G418, and 450 μm monothioglycerol (Sigma). At day 12, the EBs were transferred to tissue culture plates for adherent growth and differentiation in a defined MC medium (DMEM containing 10% FCS, 2 mm l-glutamine, 50 μg/ml G418, and 450 μm monothioglycerol (Sigma), 50 ng/ml rmSCF (Peprotech), and 30% WEHI-3 cell conditioned medium as an IL-3 supplement. Once a week, half of the medium containing the newly developed MCs was removed from the dishes, and new medium was added. Transferred nonadherent cells were grown continuously in MC medium. Starting from 2 weeks, an aliquot was checked weekly by May-Grünwald/Giemsa for MC morphology. The derived MCs were used for different experiments from week 5 to 9 after the initiation of MC differentiation. RNA purification from MCs, ES cells, and mouse 11-day embryo total RNA (Clontech) was performed with the E.Z.N.A. total RNA kit (Omega). The amount of RNA obtained was estimated based on absorbance at 260 nm. One μg of RNA was used as template for cDNA synthesis using SuperscriptTM II (Invitrogen,) reverse transcriptase with random hexamers. The Bio-Rad MiniOpticon system together with the Bio-Rad enzyme mix was used for quantitative PCR analysis. cDNA was amplified using the following primers: NDST1 (226 bp) forward, 5′-CCA CAA CTA TCA CAA AGG CAT CG-3′ and reverse, 5′-GAA AGG TTG ACT TTA GGG CCA C-3′; NDST2 (240 bp) forward, 5′-GTG TGG CAG AAT CCC TGT G-3′ and reverse, 5′-GTG CAG GCT CAG GAA GAA GT-3′; NDST3 (143 bp) forward, 5′-GGA GCT CTT CTT CAC TGT GGT T-3′ and reverse, 5′-TCT GAA GAC GCA GGT TGG T-3′; NDST4 (170 bp) forward, 5′-GGA GAA AAC CTG TGA CCA TTT AC-3′ and reverse, 5′-CCT TGT GAT AGT TGT TGC CAT TA-3′. The PCR was performed by denaturating the DNA at 95 °C for 10 min before amplification for 40 cycles, each cycle consisting of 95 °C, 30 s; 60 °C, 30 s; 72 °C, 30 s. Approximately 1 × 106 MCs were solubilized in 100 μl of 1 × SDS-PAGE sample buffer containing 5% β-mercaptoethanol. Equal volumes of the cell extracts were subjected to SDS-PAGE on 12% gels. The separated proteins were then blotted onto nitrocellulose membranes and blocked with 5% milk powder in TBS/0.1% Tween 20 for 1 h at room temperature. Following blocking, the membranes were incubated with antisera against carboxypeptidase A3 (CPA3), the chymase MCPT5 (also designated mMCP-5), and the tryptase MCPT6 (also designated mMCP-6), diluted 1:2,000 in TBS/2% BSA/0.1% Tween 20, at 40 °C overnight (the antisera were kindly provided by Dr. Lars Hellman, Uppsala University). Membranes were then washed extensively with TBS/0.1% Tween 20, and the secondary anti-rabbit antibody conjugated with horseradish peroxidase was left to bind for 1 h. After extensive washing, the blots were developed using a Bio-Rad detection system. 5 × 106 MCs of relevant genotypes, cultured in the MC-inducing medium described above, were metabolically labeled overnight with 200 μCi of carrier-free [35S]sulfate (Amersham Biosciences). After incubation, cells were pelleted by centrifugation for 10 min at 300 × g, washed with cold PBS, incubated in 2 ml of solubilization buffer (50 mm Tris-HCl, pH 7.5, 1% Triton X-100, 0.10 m NaCl), and centrifuged at 800 × g for 15 min. The supernatant containing radiolabeled macromolecules was recovered, and 35S-labeled glycosaminoglycans were isolated from the solubilized cell lysate on a 0.3-ml column of DEAE-Sephacel (Amersham Biosciences), equilibrated with 50 mm Tris-HCl, pH 7.4, 0.1% Triton X-100, 0.10 m NaCl. After washing the column with equilibration buffer followed by a second washing step with 50 mm acetate buffer, pH 4.0, containing 0.1% Triton X-100 and 0.10 m NaCl, the 35S-labeled glycosaminoglycans were eluted with 50 mm acetate buffer, pH 4.0, containing 0.1% Triton X-100 and 2 m NaCl. A portion of the eluted 35S-labeled glycosaminoglycans was treated with alkali (0.5 m NaOH) as described previously (39Bengtsson J. Eriksson I. Kjellén L. Biochemistry. 2003; 42: 2110-2115Crossref PubMed Scopus (21) Google Scholar). After desalting in water on PD10 columns (Amersham Biosciences), followed by lyophilization, the 35S-labeled glycosaminoglycan chains were subjected to digestion with 0.1 unit of chondroitinase ABC (Seikagaku) as described previously (40Cheung W.F. Eriksson I. Kusche-Gullberg M. Lindhal U. Kjellén L. Biochemistry. 1996; 35: 5250-5256Crossref PubMed Scopus (49) Google Scholar) or treated with nitrous acid at pH 1.5 (41Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3943-3950Crossref PubMed Scopus (83) Google Scholar). Untreated and treated 35S-labeled glycosaminoglycans were then analyzed by gel chromatography on Sephadex G50 eluted with 0.2 m NH4HCO3. Cells (10 × 106) were dissolved in 0.5 ml of Pronase buffer (1% Triton X-100, 50 mm Tris-HCl, pH 8.0, 1 mm CaCl2, 0.8 mg/ml Pronase) and incubated end-over-end for 19 h at 55 °C. After heat inactivation of the enzyme for 5 min at 96 °C, MgCl2 was added to a final concentration of 2 mm. After addition of Benzonase (12 milliunits), the sample was incubated for 2 h at 37 °C followed by heat inactivation for 5 min at 96 °C. The NaCl concentration was then adjusted to 0.1 m, and the sample was centrifuged at 13,000 × g for 10 min. The supernatant was diluted with 0.5 ml of 50 mm Tris-HCl, pH 8.0, 0.1 m NaCl, and applied to a Sep-Pak® C18 cartridge (Waters) that had been primed first with methanol, then with water, and finally with 50 mm Tris-HCl, pH 8.0, 0.1 m NaCl. The cartridge was washed with 2 ml of the Tris-HCl buffer, and the washing fraction was combined with the nonbinding fraction and used for purification of glycosaminoglycans. The sample was applied to a 0.2-ml DEAE-Sephacel column equilibrated in loading buffer (50 mm Tris-HCl, pH 8, 0.1 m NaCl, 0.1% Triton X-100). After washing with 6 column volumes of loading buffer, 6 volumes of low pH buffer (50 mm sodium acetate, pH 4.0, 0.1 m NaCl, 0.1% Triton X-100), and 6 volumes of loading buffer without Triton X-100, the glycosaminoglycans were eluted with 0.6 ml of elution buffer (50 mm Tris-HCl, pH 8.0, 1.5 m NaCl). After desalting on an NAP-10 column (Amersham Biosciences) equilibrated in water, the glycosaminoglycans were dried by SpeedVac centrifugation. The glycosaminoglycan pool was then digested with 50 milliunits of chondroitinase ABC in 100 μl of 40 mm Tris acetate buffer, pH 8.0. The digestion was allowed to proceed for 4 h at 37 °C, and the sample was then boiled to stop the reaction. After removal of 10 μl for chondroitin sulfate analysis by RPIP-HPLC, as described previously (20Ledin J. Staatz W. Li J.P. Götte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), heparin/HS was recovered after a second round of DEAE-Sephacel chromatography, as described for total glycosaminoglycan isolation. Purified heparin/HS was dissolved in 200 μl of heparinase buffer (5 mm Hepes buffer, pH 7.0, 50 mm NaCl, 1 mm CaCl2) and divided into two equal aliquots. One of the aliquots was treated with 0.4 milliunit each of heparinases I, II, and III and incubated for 16 h at 37 °C. The other aliquot (control sample) was incubated under the same conditions without enzymes. After heat inactivation for 5 min at 96 °C, the samples were analyzed by RPIP-HPLC (20Ledin J. Staatz W. Li J.P. Götte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Statistical analyses were performed using Prism 5.0 (GraphPad Software, San Diego, CA). Significant differences were determined using an unpaired t test. Results are presented as mean ± S.E. and calculated p values indicated as not significant (ns) p > 0.05 or significant *, p < 0.05. Using an in vitro cultivation protocol (36Vial D. Oliver C. Jamur M.C. Pastor M.V. da Silva Trindade E. Berenstein E. Zhang J. Siraganian R.P. J. Immunol. 2003; 171: 6178-6186Crossref PubMed Scopus (12) Google Scholar), embryos obtained from NDST1+/− and NDST1+/−2−/− intercrosses, isolated at embryonic (E) stages E10.5 to E12.5, were used as a starting point for differentiation of embryo-derived MCs. Because NDST1−/−2−/− embryos do not survive beyond E5.5, this genotype was not represented among the cultures. The embryos were placed into 24-well plates, and after 2 days of in vitro cultivation the embryo tissues were dispersed by trypsinization, and the cells were left to adhere again. Approximately 4 days to 1 week after readherence, MC differentiation was observed in all genotypes tested. No major morphological differences could be seen between cells of the different genotypes except for the presence of some “empty” vacuoles in the cells lacking NDST2 (Fig. 1A). Analysis of the MC proteases at the protein level demonstrated that the WT embryo-derived MCs resemble true connective tissue type MCs and express CPA3 as well as high levels of the chymase MCPT5 and the tryptase MCPT6 (Fig. 1B). In agreement with previous work (32Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellén L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (403) Google Scholar, 33Humphries D.E. Wong G.W. Friend D.S. Gurish M.F. Qiu W.T. Huang C. Sharpe A.H. Stevens R.L. Nature. 1999; 400: 769-772Crossref PubMed Scopus (358) Google Scholar), cells lacking NDST2, the NDST2−/−, and NDST1+/−2−/− embryo-derived MCs were devoid of activated CPA3 and MCPT5 and contained reduced levels of MCPT6 (Fig. 1B). Remarkably, NDST1−/− as well as NDST1+/− MCs seemed to store higher amounts of CPA3 and MCPT5 than WT MCs (Fig. 1B). Characterization of the glycosaminoglycans produced by the MCs in the presence of [35S]sulfate also gave interesting results. Compared with WT MCs where only 4% of the total 35S-labeled glycosaminoglycans resisted degradation with chondroitinase ABC and thus was identified as heparin/HS, ∼30% of the glycosaminoglycans produced by NDST1−/− and NDST1+/− cells was heparin/HS (Fig. 2, A–C). Analysis with ion exchange chromatography of the chondroitinase ABC-resistant 35S-glycosaminoglycans (representing HS and/or heparin) demonstrated that the NDST2-deficient cells produced HS with a lower charge density (Fig. 2D). In contrast, 35S-labeled heparin/HS produced by NDST1−/− and NDST1+/− MCs were eluted at higher ionic strength and were more homogeneous than WT heparin/HS, which in addition contained some material of lower charge density. To characterize in more detail the structure of the heparin/HS produced by the embryo-derived MCs, glycosaminoglycans from the cells were analyzed by RPIP-HPLC (20Ledin J. Staatz W. Li J.P. Götte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Glycosaminoglycans from four WT, six NDST1+/−, and two NDST1−/− MC cultures were separately analyzed (Fig. 3 and supplemental Fig. 1). Confirming the results obtained with ion exchange chromatography (Fig. 2D), the total sulfation of heparin/HS isolated from NDST1+/− and NDST1−/− cells is higher than that of heparin/HS from WT cells. Importantly, N-sulfation increased with ≈10%. Because the 2- and 6-O-sulfotransferases are largely dependent on N-sulfation for substrate recognition, an increase in O-sulfation would be expected. This was also the cas" @default.
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• W2068208889 cites W1519269060 @default.
• W2068208889 cites W1564050074 @default.
• W2068208889 cites W1599496006 @default.
• W2068208889 cites W1963522254 @default.
• W2068208889 cites W1964026030 @default.
• W2068208889 cites W1965214377 @default.
• W2068208889 cites W1966697925 @default.
• W2068208889 cites W1971246306 @default.
• W2068208889 cites W1971540677 @default.
• W2068208889 cites W1975366109 @default.
• W2068208889 cites W1976962628 @default.
• W2068208889 cites W1987536193 @default.
• W2068208889 cites W2010199873 @default.
• W2068208889 cites W2013965967 @default.
• W2068208889 cites W2017952250 @default.
• W2068208889 cites W2019462713 @default.
• W2068208889 cites W2029719975 @default.
• W2068208889 cites W2035107451 @default.
• W2068208889 cites W2036607811 @default.
• W2068208889 cites W2038018925 @default.
• W2068208889 cites W2043484559 @default.
• W2068208889 cites W2046737902 @default.
• W2068208889 cites W2064237121 @default.
• W2068208889 cites W2070318120 @default.
• W2068208889 cites W2071744608 @default.
• W2068208889 cites W2074987771 @default.
• W2068208889 cites W2075630737 @default.
• W2068208889 cites W2075712840 @default.
• W2068208889 cites W2076202143 @default.
• W2068208889 cites W2078657338 @default.
• W2068208889 cites W2082541916 @default.
• W2068208889 cites W2085236613 @default.
• W2068208889 cites W2089456472 @default.
• W2068208889 cites W2095058781 @default.
• W2068208889 cites W2095373191 @default.
• W2068208889 cites W2097120145 @default.
• W2068208889 cites W2102091298 @default.
• W2068208889 cites W2108745131 @default.
• W2068208889 cites W2122389073 @default.
• W2068208889 cites W2128584428 @default.
• W2068208889 cites W2130094155 @default.
• W2068208889 cites W2132014421 @default.
• W2068208889 cites W2132493358 @default.
• W2068208889 cites W2144471857 @default.
• W2068208889 cites W2156014566 @default.
• W2068208889 cites W2157280645 @default.
• W2068208889 cites W4251006690 @default.
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