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Sulf Loss Influences N-, 2-O-, and 6-O-Sulfation of Multiple Heparan Sulfate Proteoglycans and Modulates Fibroblast Growth Factor Signaling

2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês

10.1074/jbc.m802130200

ISSN

1083-351X

Autores

William C. Lamanna, Marc‐André Frese, Martina Balleininger, Thomas Dierks,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes. Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes. Heparan sulfate proteoglycans (HSPGs) 2The abbreviations used are: HSPG, heparan sulfate proteoglycan; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; FGF, fibroblast growth factor; GlcNS, N-sulfoglucosamine; GPI, glycosylphosphatidylinositol; HS, heparan sulfate; MEF, mouse embryonic fibroblast; 2S, 2-O-sulfation; 6S, 6-O-sulfation; PI-PLC, phosphatidylinositol-phospholipase C; HPLC, high performance liquid chromatography; UA, hexuronic acid; ΔUA, Δ4,5-unsaturated hexuronic acid; WT, wild type; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting. are essential regulators of cell signaling and development that are ubiquitously present on the cell surface and in the extracellular matrix (ECM) of virtually all animal cells (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2332) Google Scholar). Composed of a dynamically sulfated heparan sulfate (HS) polymer and a protein backbone, HSPGs can be divided into three functionally distinct families: the transmembrane syndecans, the glycosylphosphatidylinositol (GPI)-anchored glypicans, and the ECM-associated proteoglycans perlecan, agrin, and collagen XVIII (2Bulow H.E. Hobert O. Annu. Rev. Cell Dev. Biol. 2006; 22: 375-407Crossref PubMed Scopus (275) Google Scholar). A major function of the HS component of HSPGs is to act as a regulatory cofactor for a variety of signaling molecules and morphogens (3Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1292) Google Scholar, 4Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2094) Google Scholar, 5Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 6Reichsman F. Smith L. Cumberledge S. J. Cell Biol. 1996; 135: 819-827Crossref PubMed Scopus (261) Google Scholar). In addition to the HS chains, the protein backbone of HSPGs also plays a critical role in determining proteoglycan function, regulating processes such as cellular adhesion, HSPG shedding, and endocytosis (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2332) Google Scholar, 7Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (337) Google Scholar, 8Zimmermann P. Zhang Z. Degeest G. Mortier E. Leenaerts I. Coomans C. Schulz J. N′Kuli F. Courtoy P.J. David G. Dev. Cell. 2005; 9: 377-388Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). HSPG biosynthesis is a non-template-driven process, relying on multiple enzyme activities to generate a glycosaminoglycan polymer with distinct sulfation patterns attached to a protein core. Synthesis begins with the attachment of a tetrasaccharide linker sequence onto a target serine residue, followed by polymerization of alternating GlcA and GlcNAc residues up to 200 disaccharides in length. Once synthesized, the diversity of HS structure is generated by a variety of heparan-modifying enzymes in the Golgi, which epimerize a portion of the GlcA residues to iduronic acid and add sulfate to some of the 2-O-positions of the hexuronic acid (UA) and N- and 6-O-positions of GlcNAc (9Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1254) Google Scholar). Importantly, the cumulative action of these biosynthetic enzymes is incomplete, generating a defined HS domain structure composed of highly (S-domains), partially (transition zones), and nonsulfated regions (10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). These patterns of sulfation are cell type- and developmental stage-specific (11Ledin J. Staatz W. Li J.P. Gotte M. Selleck S. Kjellen L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), serving as dynamic templates to promote or inhibit specific cellular interactions and signaling events (12Gallagher J.T. Biochem. Soc. Trans. 2006; 34: 438-441Crossref PubMed Scopus (58) Google Scholar, 13Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (400) Google Scholar). In recent years, the activity of two additional HS-modifying enzymes has been described, Sulf1 and Sulf2 (14Dhoot G.K. Gustafsson M.K. Ai X. Sun W. Standiford D.M. Emerson Jr., C.P. Science. 2001; 293: 1663-1666Crossref PubMed Scopus (391) Google Scholar, 15Morimoto-Tomita M. Uchimura K. Werb Z. Hemmerich S. Rosen S.D. J. Biol. Chem. 2002; 277: 49175-49185Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). These heparan sulfate 6-O-endosulfatases are unique in their ability to postsynthetically edit 6-O-sulfation patterns at the cell surface. Experiments exploiting in vitro or Sulf-overexpressing systems have demonstrated that enzymatic removal of 6-O-sulfate by the Sulfs has significant effects on the activity of a number of growth factors and morphogens, such as FGF, Wnt, bone morphogenetic protein, and vascular endothelial growth factor (16Ai X. Do A.T. Lozynska O. Kusche-Gullberg M. Lindahl U. Emerson Jr., C.P. J. Cell Biol. 2003; 162: 341-351Crossref PubMed Scopus (369) Google Scholar, 17Viviano B.L. Paine-Saunders S. Gasiunas N. Gallagher J. Saunders S. J. Biol. Chem. 2004; 279: 5604-5611Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 18Wang S. Ai X. Freeman S.D. Pownall M.E. Lu Q. Kessler D.S. Emerson Jr., C.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4833-4838Crossref PubMed Scopus (183) Google Scholar, 19Narita K. Staub J. Chien J. Meyer K. Bauer M. Friedl A. Ramakrishnan S. Shridhar V. Cancer Res. 2006; 66: 6025-6032Crossref PubMed Scopus (117) Google Scholar, 20Uchimura K. Morimoto-Tomita M. Bistrup A. Li J. Lyon M. Gallagher J. Werb Z. Rosen S.D. BMC Biochem. 2006; 7: 2Crossref PubMed Scopus (186) Google Scholar). In order to understand the biological significance of these enzymes during mammalian development, knock-out mouse models were generated and subsequently characterized. Although loss of Sulf1 or Sulf2 was shown to result in relatively mild phenotypes variably affecting lung development as well as growth and viability, the loss of both Sulfs resulted in severe developmental ablations and early post-natal lethality (21Ai X. Kitazawa T. Do A.T. Kusche-Gullberg M. Labosky P.A. Emerson Jr., C.P. Development. 2007; 134: 3327-3338Crossref PubMed Scopus (143) Google Scholar, 22Holst C.R. Bou-Reslan H. Gore B.B. Wong K. Grant D. Chalasani S. Carano R.A. Frantz G.D. Tessier-Lavigne M. Bolon B. French D.M. Ashkenazi A. PLoS ONE. 2007; 2: e575Crossref PubMed Scopus (101) Google Scholar, 23Lamanna W.C. Kalus I. Padva M. Baldwin R.J. Merry C.L. Dierks T. J. Biotechnol. 2007; 129: 290-307Crossref PubMed Scopus (143) Google Scholar, 24Lum D.H. Tan J. Rosen S.D. Werb Z. Mol. Cell. Biol. 2007; 27: 678-688Crossref PubMed Scopus (77) Google Scholar, 25Ratzka A. Kalus I. Moser M. Dierks T. Mundlos S. Vortkamp A. Dev. Dyn. 2008; 237: 339-353Crossref PubMed Scopus (72) Google Scholar). These phenotypic observations, in conjunction with previous analyses of HS material from Sulf knock-out mouse embryonic fibroblasts (MEFs), support the idea that the Sulfs act cooperatively in vivo to modify HS sulfation patterns and regulate developmental processes (26Lamanna W.C. Baldwin R.J. Padva M. Kalus I. Ten Dam G. van Kuppevelt T.H. Gallagher J.T. von Figura K. Dierks T. Merry C.L. Biochem. J. 2006; 400: 63-73Crossref PubMed Scopus (110) Google Scholar). Despite the biological importance of the Sulfs, much remains to be learned about the endogenous activity and HSPG substrate specificity of these enzymes. Indeed, primary Sulf knock-out MEFs have been shown to exhibit changes in multiple sulfate groups not accounted for by in vitro Sulf activity assays employing heparin substrate analogs (15Morimoto-Tomita M. Uchimura K. Werb Z. Hemmerich S. Rosen S.D. J. Biol. Chem. 2002; 277: 49175-49185Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 26Lamanna W.C. Baldwin R.J. Padva M. Kalus I. Ten Dam G. van Kuppevelt T.H. Gallagher J.T. von Figura K. Dierks T. Merry C.L. Biochem. J. 2006; 400: 63-73Crossref PubMed Scopus (110) Google Scholar, 27Saad O.M. Ebel H. Uchimura K. Rosen S.D. Bertozzi C.R. Leary J.A. Glycobiology. 2005; 15: 818-826Crossref PubMed Scopus (86) Google Scholar). Further, inconsistent results regarding the ability of the Sulfs to modify HSPG substrates within the ECM have been reported (28Dai Y. Yang Y. MacLeod V. Yue X. Rapraeger A.C. Shriver Z. Venkataraman G. Sasisekharan R. Sanderson R.D. J. Biol. Chem. 2005; 280: 40066-40073Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 29Ai X. Do A.T. Kusche-Gullberg M. Lindahl U. Lu K. Emerson Jr., C.P. J. Biol. Chem. 2006; 281: 4969-4976Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). To address these issues, in vitro activity analysis using native HS substrates was carried out in conjunction with the characterization of cell surface, GPI-anchored, shed, and ECM-associated HSPGs from immortalized WT and Sulf knock-out cell lines. These studies demonstrate a highly restricted 6-O-sulfate substrate specificity for the Sulfs in vitro, which was contrasted by dynamic effects of Sulf loss on N-, 2-O-, and 6-O-sulfated moieties in vivo. This comparative analysis implicates that, in addition to their 6-O-endosulfatase activity, Sulf1 and Sulf2 are able to dynamically modulate the abundance of nonsubstrate N-, 2-O-, and 6-O-sulfate groups on proteoglycans throughout the cell surface and the ECM by influencing the HS biosynthetic machinery. In support of this novel observation, real time PCR expression analysis was able to verify a significant impact of Sulf loss on the abundance of HS sulfotransferase transcripts. Finally, the modulation of HS sulfation patterns resulting from Sulf deficiency observed in this study was shown to elicit significant effects on FGF signaling, underlining the importance of these enzymes in regulating cellular response. Materials—Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were from Invitrogen and Lonza, respectively. Heparinases I–III (from Flavobacterium heparinum) were from Grampian Enzymes (Orkney, UK). Chondroitinase ABC (from Proteus vulgaris) was from Sigma. Phosphatidylinositol-phospholipase C (from Bacillus cereus) was from Invitrogen. Antibodies directed against nonphosphorylated and diphosphorylated ERK1/2 were from Sigma. Anti-RGS-His6 antibodies were from Qiagen. The rat anti-perlecan antibodies were from Chemicon International. Anti-GM130 antibodies were from BD Transduction Laboratories. The monoclonal anti-HS stub antibody 3G10 was a kind gift from Professor Guido David (University of Leuven, Belgium). Antibodies against native Sulf1 and Sulf2 were provided by Shire Human Genetic Therapies Inc. (Cambridge, MA). Chromatography gels used were Sepharose CL-6B (GE Healthcare), P2 extra fine (Bio-Rad), DEAE-Sephacel (Sigma), and nickel-Sepharose 6 Fast Flow (GE Healthcare). The ProPac PA-1 HPLC column was from Dionex (Camberley, UK). d-[6-3H]-glucosamine hydrochloride and Na2[35S]O4 were from PerkinElmer Life Sciences. Recombinant human FGF2 was from R&D Systems. MEF Immortalization and Radioactive Labeling of HS Chains—Generation of Sulf knock-out mice has been described previously (26Lamanna W.C. Baldwin R.J. Padva M. Kalus I. Ten Dam G. van Kuppevelt T.H. Gallagher J.T. von Figura K. Dierks T. Merry C.L. Biochem. J. 2006; 400: 63-73Crossref PubMed Scopus (110) Google Scholar). MEF cultures of WT, Sulf1-/-, Sulf2-/-, or Sulf1-/-/Sulf2-/- genotypes (embryonic day 12.5) were immortalized via stable transfection with the pMSSVLT vector containing the SV40 large T gene (30Schuermann M. Nucleic Acids Res. 1990; 18: 4945-4946Crossref PubMed Scopus (33) Google Scholar). For radioactive labeling, immortalized MEF cell lines were cultured in d-[6-3H]glucosamine hydrochloride (50 μCi/ml) and Na2[35S]O4 (50 μCi/ml) for 48 h, as previously described (31Merry C.L. Bullock S.L. Swan D.C. Backen A.C. Lyon M. Beddington R.S. Wilson V.A. Gallagher J.T. J. Biol. Chem. 2001; 276: 35429-35434Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), prior to the proteoglycan fractionation described below. Cell Surface and Shed Proteoglycan Fractionation—Conditioned medium was collected after 48 h, cleared of cellular debris via centrifugation, and pooled as the shed proteoglycan fraction. Cells were subsequently washed twice in PBS and treated for 10 min with trypsin/EDTA solution (Invitrogen). Cells were pelleted and washed twice in HEPES, 0.9% NaCl, 0.5 mm EDTA, and supernatant samples were pooled together as the cell surface proteoglycan fraction. GPI-anchored Proteoglycan Fractionation—The protocol for GPI-anchored glypican enrichment and purification was based on the protocol of Tumova et al. (32Tumova S. Woods A. Couchman J.R. J. Biol. Chem. 2000; 275: 9410-9417Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, cells were washed three times with PBS and incubated for 45 min at 37 °C in serum-free DMEM containing 0.067 units/ml phosphatidylinositol-phospholipase C (PI-PLC). Supernatant was collected, and cells were washed twice with 10 mm HEPES, 0.9% NaCl, 0.5 mm EDTA. These wash fractions were pooled with the supernatant as the glypican proteoglycan fraction. ECM Proteoglycan Fractionation—MEF cell lines were plated onto 15-cm sterile glass Petri dishes coated with poly-l-lysine (Sigma) and 1 μg/ml laminin (Sigma). Lipid extraction was carried out based on previous experiments by Heremans et al. (33Heremans A. Cassiman J.J. Van den Berghe H. David G. J. Biol. Chem. 1988; 263: 4731-4739Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were washed twice with ice-cold PBS, washed three times for 5 min with 0.5% Triton X-100 in 10 mm Tris/HCl, 150 mm NaCl, 10 mm EDTA, pH 8.0, and washed three times for 10 min with 0.5% sodium deoxycholate in 10 mm Tris, 150 mm NaCl, pH 8.0. The matrix that remained attached to the glass plates was stringently washed three times with 0.2 m Tris, 2 m NaCl, pH 8.0, before finally being washed three times with 0.2 m Tris, pH 8.0. ECM was harvested in PBS containing collagenase II (10 units/ml) (PAA Laboratories, Pasching, Austria) and incubated overnight at 37 °C. Disaccharide Composition Analysis—Purified HS chains were digested with a combination of heparinases I, II, and III, and the resultant disaccharides were identified following strong anion exchange-HPLC as previously described (31Merry C.L. Bullock S.L. Swan D.C. Backen A.C. Lyon M. Beddington R.S. Wilson V.A. Gallagher J.T. J. Biol. Chem. 2001; 276: 35429-35434Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Cell Surface Proteoglycan Profiling—Cells were grown to 100% confluence on a 10-cm plate and were washed twice with ice-cold PBS, harvested, pelleted, and resuspended in 50 mm HEPES, pH 8.0. Cells were homogenized manually with a Dounce homogenizer. Cellular debris was cleared by centrifuging at 500 × g for 5 min at 4 °C, and the supernatant was centrifuged at 100,000 × g for 1 h at 4 °C to pellet the membrane fraction. Cell membranes were resuspended in 50 mm HEPES containing 0.5% Triton X-100, pH 8.0, and incubated on ice for 30 min. The sample was centrifuged at 100,000 × g for 30 min, and the Triton-soluble supernatant was collected, diluted to 0.1% Triton with 50 mm sodium acetate, 0.5 mm calcium acetate, pH 7.0, and digested for 2 h at 37 °C with 2 mIU heparinase II and 2 mIU heparinase III. GPI-anchored Proteoglycan Profiling—Cells were grown to 100% confluence on a 10-cm plate, and GPI-anchored glypican was extracted as described above. Following PI-PLC treatment, conditioned medium was bound to a 1-ml HiTrap DEAE FF column (GE Healthcare), washed with 20 ml of PBS, and eluted with PBS containing 1.5 m NaCl. Protein-containing fractions were pooled, dialyzed against 50 mm sodium acetate, 0.5 mm calcium acetate, pH 7.0, and digested for 2 h at 37 °C with 2 mIU heparinase II and 2 mIU heparinase III. Shed Proteoglycan Profiling—Cells were grown to 100% confluence on a 10-cm plate, washed twice with PBS, and incubated for 24 h in serum-free DMEM. Conditioned medium was bound to a 1-ml HiTrap DEAE FF column (GE Healthcare), washed with 50 ml of PBS, and eluted with PBS containing 1.5 m NaCl. Samples were dialyzed and digested with heparinases II and III, as described above. ECM Proteoglycan Profiling—Confluent cells plated on glass coverslips in a 24-well dish were treated for 2 h with 2 mIU heparinase II and 2 mIU heparinase III in serum-free DMEM followed by ECM extraction, as described above. ECM composition was analyzed via immunofluorescence microscopy according to a standard fixation and staining protocol as previously described (34Frese M.A. Schulz S. Dierks T. J. Biol. Chem. 2008; 283: 11388-11395Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Alternatively, the ECM was scraped from the glass coverslips for Western blotting. Cloning of Human Sulf1 and Sulf2 cDNAs and Construction of Expression Vectors—A 5′-truncated Sulf2 cDNA, designated KIAA1247 (35Nagase T. Ishikawa K. Kikuno R. Hirosawa M. Nomura N. Ohara O. DNA Res. 1999; 6: 337-345Crossref PubMed Scopus (119) Google Scholar), was obtained from the Kazusa Institute (Kisarazu, Chiba, Japan). Using primer 1247_His, a RGS-His6 tag-encoding sequence, followed by a HindIII site, was added to the 3′-end of the coding region by PCR (for PCR primers, see Table S1). The product was subcloned as a 1.8-kb EcoRI/HindIII fragment into vector pMPSVEH (36Artelt P. Morelle C. Ausmeier M. Fitzek M. Hauser H. Gene (Amst.). 1988; 68: 213-219Crossref PubMed Scopus (134) Google Scholar). The lacking 5′ region of the Sulf2 cDNA was obtained through rapid amplification of cDNA ends-PCR using the GeneRacer kit (Invitrogen). As gene-specific noncoding primers, 1247_SP3 and, for nested PCR, 1247_SP4 were used. From the obtained 1.0-kb product (Sulf2-N), the 5′-untranslated region was deleted by adding a 5′-NcoI site at the position of the start codon (primer 1247_NcoI), which allowed fusion with the optimized Kozak sequence of a pMPSVEH-based arylsulfatase A expression vector described earlier (37Dierks T. Schmidt B. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11963-11968Crossref PubMed Scopus (111) Google Scholar). Full-length Sulf2-RGS-His6-encoding cDNA was obtained by joining the 5′ and 3′ fragments via the internal EcoRI site, which was amplified also with the rapid amplification of cDNA ends product. For construction of the pCI-neo-based expression vector, the 3′ Sulf2-RGS-His6 fragment was cloned first as an EcoRI/HindIII fragment (blunted at the HindIII end) into pCI-neo (Promega), which had been opened by EcoRI and SmaI, and then assembled with the 5′ EcoRI/EcoRI fragment excised from the pMPSV-Kozak-Sulf2-N construct. Cloning of Sulf1 cDNA also started from a 5′-truncated EST fragment (KIAA1077), which was obtained from the Kazusa Institute (38Kikuno R. Nagase T. Ishikawa K. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1999; 6: 197-205Crossref PubMed Scopus (175) Google Scholar). Using primer 1077_His, an RGS-His6 tag encoding sequence was added to the 3′-end of the coding region, followed by an MscI site. The obtained 2.3-kb product (Sulf1-C) was subcloned as an AccI/MscI fragment. The lacking 5′ region of Sulf1 cDNA was obtained through rapid amplification of cDNA ends-PCR using the gene-specific noncoding primer 1077_SP3. From the obtained 0.4-kb Sulf1-N product, the 5′-untranslated region was deleted by adding a 5′ NcoI site at the position of the start codon (primer 1077_NcoI), which allowed generation of an optimized Kozak sequence (as above). Full-length Sulf1-RGS-His6-encoding cDNA was obtained by joining 5′ and 3′ fragments via the internal AccI site. For that purpose, Kozak-Sulf1-N was cloned as an EcoRI/AccI fragment into the MCS of pCI-neo and then assembled with Sulf1-C as an AccI/AccI fragment. Full-length sequencing resulted in the same coding sequences for both Sulf1 and Sulf2 as published by Morimoto-Tomita et al. (15Morimoto-Tomita M. Uchimura K. Werb Z. Hemmerich S. Rosen S.D. J. Biol. Chem. 2002; 277: 49175-49185Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). To generate plasmids with Sulf cDNAs encoding enzymatically inactive Sulf1-C87A/C88A or Sulf2-C88A/C89A mutants, the QuikChange method (Stratagene) was applied, using complementary mutagenesis primers Sulf1CA or Sulf2CA (each forward and reverse), respectively (for PCR primers see Table S2). Expression and Localization Analysis of Human Sulfs in HT1080 Cells—Human fibrosarcoma HT1080 cells were transfected with pCI-neo plasmids containing wild type or mutant Sulf cDNAs using magnet-assisted transfection according to the manufacturer's instructions (IBA, Göttingen, Germany). Stable clones were selected with 800 μg/ml G-418 sulfate (PAA Laboratories), and drug-resistant cells were cloned and expanded. Sulf expression in total lysates was analyzed by lysing cells with gentle sonification in 10 mm HEPES, 0.5 m NaCl, pH 7.4, centrifugation at 100,000 × g for 30 min at 4 °C, and Western blotting using anti-RGS-His6 antibodies. Detergent solubility was tested by lysing cells manually with a cell Dounce homogenizer in 50 mm HEPES, pH 8.0, centrifuging 500 × g for 5 min at 4 °C to remove cellular debris and subsequently centrifuging the supernatant at 100,000 × g for 1 h at 4 °C to isolate the membrane fraction. Membranes were treated with 1 or 2.5% Triton X-100 or Brij98 on ice for 30 min prior to centrifugation at 100,000 × g for 1 h at 4 °C to separate detergent-soluble and detergent-insoluble fractions. For detection of secreted Sulfs in Western blots, confluent Sulf-expressing cell lines were cultivated for 72 h in serum-free DMEM prior to 100× concentration of conditioned medium using Centricon-30 units (Millipore), as previously described (15Morimoto-Tomita M. Uchimura K. Werb Z. Hemmerich S. Rosen S.D. J. Biol. Chem. 2002; 277: 49175-49185Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Immunofluorescence analysis of permeabilized HT1080 cells expressing Sulf1 or Sulf2 was performed as previously described (34Frese M.A. Schulz S. Dierks T. J. Biol. Chem. 2008; 283: 11388-11395Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) but without LysoTracker treatment. Alternatively, live cells were stained to specifically label cell surface-localized proteins. Sulf-expressing HT1080 cells were incubated for 1 h at 4 °C with anti-RGS-His6 antibodies in serum-free DMEM, washed with PBS, and fixed with 4% paraformaldehyde. After blocking with 2% fetal calf serum, the cells were labeled with an Alexa-488-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes) and imaged as previously described (34Frese M.A. Schulz S. Dierks T. J. Biol. Chem. 2008; 283: 11388-11395Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In Vitro Sulf Activity Analysis—For in vitro analysis of Sulf activity from cell lysates, Sulf-expressing cells were harvested, pelleted, and lysed with gentle sonification in 20 mm Tris, 0.5 m NaCl, 10 mm imidazole, pH 7.4. Cleared cell lysate was added to 100 μl of nickel-Sepharose and incubated at 4 °C overnight. Nickel beads were washed three times with 20 mm Tris, 0.5 m NaCl, 40 mm imidazole, pH 7.4, and finally twice in 50 mm Tris, 10 mm MgCl2, pH 7.4, activity buffer. Nickel-bound Sulfs were incubated overnight at 37 °C in activity buffer containing 200,000 3H cpm of purified HS isolated from Sulf1/2 double knock-out MEFs. For analysis of secreted Sulf activity, cells were cultivated in DMEM with 10% fetal calf serum for 72 h prior to (NH4)2SO4 precipitation of conditioned medium. Medium precipitate was resuspended and dialyzed against activity buffer. The sample volume of the dialyzed material was reduced to 10% of the starting volume using a SpeedVac and incubated overnight at 37 °C with 200,000 3H cpm of purified HS as above. HS was rebound on 500 μl of DEAE resin and washed with 20 ml of PBS to remove excess proteins prior to elution with 1.5 m NaCl. HS samples were desalted on PD10 columns (GE Healthcare), and disaccharide compositions were analyzed as previously described (31Merry C.L. Bullock S.L. Swan D.C. Backen A.C. Lyon M. Beddington R.S. Wilson V.A. Gallagher J.T. J. Biol. Chem. 2001; 276: 35429-35434Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Real Time PCR—Total RNA was extracted from MEF cell lines using the RNeasy minikit (Qiagen). Synthesis of first-strand cDNA from mRNA transcripts was performed using the iScript cDNA synthesis kit (Bio-Rad). Real time PCR was carried out using the LightCycler FastStart DNA Master (Plus) SYBR Green I Kit and the LightCycler instrument (both from Roche Applied Science). The conditions for denaturation, annealing, and extension were repeated 45 times as follows: denaturation at 95 °C for 10 s, annealing at 61 °C for 5 s, and extension at 72 °C for 10 s. All reactions were performed in triplicates. Real time PCR primers are listed in Table S3. Primer specificities were analyzed by agarose gel electrophoresis to verify amplification of single products of the expected sizes and by melting curve analysis. cDNA dilution series were performed to calculate PCR primer efficiencies. Efficiencies were 90–100% for all primer sets used. Relative quantification of transcripts was carried out according to Pfaffl (39Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25871) Google Scholar) with data normalized to the housekeeping gene Rpl13a. FACS Analysis—Cells were grown to 100% confluence, washed twice with PBS, and incubated for 2 h in

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