Artigo Acesso aberto Revisado por pares

Arylsulfatase G, a Novel Lysosomal Sulfatase

2008; Elsevier BV; Volume: 283; Issue: 17 Linguagem: Inglês

10.1074/jbc.m709917200

ISSN

1083-351X

Autores

Marc‐André Frese, S. Schulz, Thomas Dierks,

Tópico(s)

Trypanosoma species research and implications

Resumo

The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site Cα-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder. The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site Cα-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder. Sulfatases represent a family of enzymes essential for the degradation and remodeling of sulfate esters. In mammals, sulfatases are involved in the turnover of various sulfated substrates such as glycosaminoglycans (heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate), steroid hormones (e.g. dehydroepiandrosteron 3-sulfate), and sulfolipids (e.g. cerebroside-3-sulfate) (1Hopwood, J. J., and Ballabio, A. (2001) in The Molecular and Metabolic Bases of Inherited Disease (Scriver, C., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 8th Ed., Vol. 3, pp. 3725-3732, McGraw-Hill, New YorkGoogle Scholar, 2Diez-Roux G. Ballabio A. Annu. Rev. Genomics Hum. Genet. 2005; 6: 355-379Crossref PubMed Scopus (148) Google Scholar). Furthermore, they have important regulatory functions in modulating heparan sulfate-dependent cell signaling pathways (3Lamanna W.C. Kalus I. Padva M. Baldwin R.J. Merry C.L.R. Dierks T. J. Biotechnol. 2007; 129: 290-307Crossref PubMed Scopus (143) Google Scholar, 4Wang 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, 5Dhoot 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, 6Viviano B.L. Paine-Saunders S. Gasiunas N. Gallagher J. Saunders S. J. 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In vivo, sulfatases display stringent specificities toward their individual substrates and have low functional redundancy. Their active sites contain a unique Cα-formylglycine (FGly) 2The abbreviations used are: FGly, Cα-formylglycine; 4-MUS, 4-methylumbelliferyl sulfate; ASG, arylsulfatase G; ER, endoplasmic reticulum; M6PR, mannose 6-phosphate receptor; pNCS, p-nitrocatechol sulfate; pNPS, p-nitrophenyl sulfate; MES, 4-morpholineethanesulfonic acid. that is post-translationally generated in the endoplasmic reticulum by oxidation of a conserved cysteine residue (12Schmidt B. Selmer T. Ingendoh A. von Figura K. Cell. 1995; 82: 271-278Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 13Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 14Cosma M.P. Pepe S. Annunziata I. Newbold R.F. Grompe M. Parenti G. Ballabio A. Cell. 2003; 113: 445-456Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). A genetic defect of the formylglycine-generating enzyme (FGE) leads to multiple sulfatase deficiency, a rare but fatal inherited disease in which all sulfatases are catalytically inactive due to a lack of FGly (1Hopwood, J. J., and Ballabio, A. (2001) in The Molecular and Metabolic Bases of Inherited Disease (Scriver, C., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 8th Ed., Vol. 3, pp. 3725-3732, McGraw-Hill, New YorkGoogle Scholar, 13Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 14Cosma M.P. Pepe S. Annunziata I. Newbold R.F. Grompe M. Parenti G. Ballabio A. Cell. 2003; 113: 445-456Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 15Dierks T. Dickmanns A. Preusser-Kunze A. Schmidt B. Mariappan M. von Figura K. Ficner R. Rudolph M.G. Cell. 2005; 121: 541-552Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Twelve of the 17 sulfatases encoded in the human genome have been characterized biochemically (16Sardiello M. Annunziata I. Roma G. Ballabio A. Hum. Mol. Genet. 2005; 14: 3203-3217Crossref PubMed Scopus (140) Google Scholar). Based on their subcellular localization they can be divided into lysosomal and non-lysosomal enzymes. The latter are found either at the cell surface (Sulf1, Sulf2), in the endoplasmic reticulum (arylsulfatases C, D, and F), or in the Golgi apparatus (arylsulfatase E) and act at neutral pH. In contrast, all lysosomal sulfatases (arylsulfatases A and B, iduronate-2-sulfatase, heparan-N-sulfatase, glucosamine-6-sulfatase, and galactosamine-6-sulfatase) share an acidic pH optimum (17Hanson S.R. Best M.D. Wong C.H. Angew. Chem. Int. Ed. Eng. 2004; 43: 5736-5763Crossref PubMed Scopus (288) Google Scholar). The genetic deficiency of each of these six lysosomal sulfatases causes specific and severe lysosomal storage disorders, namely metachromatic leukodystrophy and mucopolysaccharidoses type VI, II, IIIA, IIID, and IVA, respectively, which highlights the essential and non-redundant function of these enzymes (2Diez-Roux G. Ballabio A. Annu. Rev. Genomics Hum. Genet. 2005; 6: 355-379Crossref PubMed Scopus (148) Google Scholar). In affected patients, the degradation of a specific sulfated compound is blocked, leading to its accumulation in the lysosomes and in the extracellular fluids. Lysosomal storage impairs autophagic delivery of bulk cytosolic contents to lysosomes, finally resulting in accumulation of toxic proteins, cellular damage, and apoptosis (18Settembre C. Fraldi A. Jahreiss L. Spampanato C. Venturi C. Medina D. de Pablo R. Tacchetti C. Rubinsztein D.C. Ballabio A. Hum. Mol. Genet. 2008; 17: 119-129Crossref PubMed Scopus (412) Google Scholar). The total number of lysosomal hydrolases, according to proteomic analyses, is estimated to be in the range of 50–60 (19Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (637) Google Scholar). In principle, a genetic mutation of any of these proteins can cause a lysosomal storage disorder. For many sulfated substrates like sulfo-proteins (tyrosine-, threonine- or serine-O-sulfate (20Medzihradszky K.F. Darula Z. Perlson Z. Fainzilber M. Chalkley R.J. Ball H. Greenbaum D. Bogyo M. Tyson D.R. Bradshaw R.A. Burlingame A.L. Mol. Cell. Proteomics. 2004; 3: 429-440Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the selectin ligand 6-sulfo sialyl LewisX, as well as the heparan sulfate constituents N-acetylglucosamine 3-sulfate and glucuronate 2-sulfate), the corresponding sulfatases and possible associated storage disorders have not been identified yet. Thus, the discovery and characterization of novel lysosomal enzymes will likely be important in the identification and molecular understanding of so far non-classified inherited genetic diseases. The mammalian arylsulfatase G (ASG) was identified in 2002 through bioinformatic searches of expressed sequence tag databases (21Ferrante P. Messali S. Meroni G. Ballabio A. Eur. J. Hum. Genet. 2002; 10: 813-818Crossref PubMed Scopus (47) Google Scholar). The ARSG gene is located on chromosome 17q24.2, consists of 11 exons, and encodes a 525-amino acid protein that shares a high degree of similarity with all sulfatases, in particular with arylsulfatase A (50% sequence similarity and 37% identity). The enzyme was tentatively classified as an arylsulfatase, although no activity toward the commonly used arylsulfate pseudosubstrates p-nitrocatechol sulfate (pNCS) and 4-methylumbelliferyl sulfate (4-MUS) could be detected. In overexpressing COS-7 cells, ASG was found to be localized in the endoplasmic reticulum. In this study, however, we demonstrate for the first time that ASG is an active arylsulfatase enzyme of the lysosome. Construction of Expression Plasmids—The full-length cDNA sequence encoding human ASG, designated KIAA1001 (accession number NM_014960.1, 22), was obtained from the Kazusa Institute (Kisarazu, Chiba, Japan). Two sequence isoforms of ASG are known. Whereas genomic and expressed sequence tag sequences, both for human and murine ASG, contain a GCA (Ala) codon in position 501, this codon is replaced by CCA (Pro) in the KIAA1001 clone (21Ferrante P. Messali S. Meroni G. Ballabio A. Eur. J. Hum. Genet. 2002; 10: 813-818Crossref PubMed Scopus (47) Google Scholar). The ASG A501P cDNA was amplified by PCR, thereby adding a 3′-RGS-His6-encoding sequence followed by a stop codon and a HindIII site (reverse primer 5′-CCCAAGCTTAGTGATGGTGATGGTGATGCGATCCTCTTGCGGCTTGACAGCGGC-3′). The PCR product was digested with HindIII and NcoI, which cuts in the endogenous Kozak sequence of ASG, and cloned into pMPSV-EH-ASA (23Dierks T. Schmidt B. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11963-11968Crossref PubMed Scopus (111) Google Scholar), thus exchanging the entire coding region of arylsulfatase A in-frame to that of ASG. The insert was then subcloned as a 5′-EcoRI-3′-HindIII/blunt fragment into the EcoRI/SmaI-opened multiple cloning site of pCI-neo (Promega). The QuikChange mutagenesis protocol (Stratagene) was used to generate the wild-type ASG-encoding sequence with a GCA codon in position 501 (accession number NM_014960.2). For this purpose, complementary primers were used (forward 5′-CGACAACATCTCCAGCGCAGATTACACTCAGG-3′, reverse 5′-CCTGAGTGTAATCTGCGCTGGAGATGTTGTCG-3′). The ASG C84A mutant was prepared accordingly (forward 5′-GCTGCCTCCACCGCCTCACCCTCCCG-3′, reverse 5′-CGGGAGGGTGAGGCGGTGGAGGCAGC-3′). All constructs were full-length sequenced in the coding region to preclude any PCR-derived errors. Unless otherwise stated, all experiments were performed with wild-type ASG. Cell Culture and Transfections—HT1080 human fibrosarcoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin/streptomycin (Invitrogen) under 5% CO2 atmosphere at 37 °C. Transfections with BamHI-linearized pCI-neo-ASG plasmids were performed using MATra-A transfection reagent (IBA, Göttingen, Germany) following the protocol recommended by the manufacturer. After growth for 12 days in medium containing 800 μg/ml G-418 sulfate (PAA Laboratories, Pasching, Austria), drug-resistant cells were cloned and expanded. Clones were screened by Western blot analysis for ASG expression. Purification from Cell Culture Supernatants—HT1080 cells stably overexpressing ASG-His were grown to near confluence in growth medium containing 10% fetal calf serum on 15-cm cell culture dishes (Nunc) or Cellmaster PS roller bottles (Greiner Bio-One). During ASG production, the amount of serum was reduced to 1%. The conditioned medium was collected every 48–72 h, cleared by spinning, and subjected to ammonium sulfate precipitation (50% w/v). The precipitate was dialyzed overnight at 4 °C against binding buffer (20 mm Tris-HCl, 500 mm NaCl, 40 mm imidazole, pH 7.4). The dialyzed material was cleared by centrifugation (18,000 × g, 3 × 30 min, 4 °C), passed through a 0.22-μm nitrocellulose filter, and bound onto a HisTrap HP 1-ml column connected to an ÄKTA Explorer 10 system (GE Healthcare). The column was washed with binding buffer, and proteins were gradually eluted with elution buffer (20 mm Tris-HCl, 500 mm NaCl, 500 mm imidazole, pH 7.4). Peak fractions, as analyzed by Western blotting and pNCS assays, were pooled, dialyzed against buffer A (20 mm MES, pH 6.0), and applied onto a RESOURCE S 1-ml cation exchange column (GE Healthcare). Elution was carried out using a gradient with buffer B (20 mm MES, 1 m NaCl, pH 6.0). The ASG-His protein eluted at 140 mm NaCl. Peak fractions were pooled and concentrated by speed vac, if necessary. Proteins were analyzed by SDS-PAGE on 15% polyacrylamide gels and stained with Roti-Blue colloidal Coomassie (Carl Roth, Karlsruhe, Germany). Protein concentrations were determined using Coomassie Plus Bradford reagent (Pierce). Average yields were ∼40 μg of purified ASG/liter of medium. Peptide N-Glycosidase F and Endoglucosaminidase H Treatment—HT1080 cells stably overexpressing ASG-His were grown to confluency. The cells were harvested by rubber policeman and extracted by sonication in lysis buffer (10 mm HEPES, 0.5 m NaCl, pH 7.4). The lysate was cleared by centrifugation (72,000 × g, 20 min, 4 °C). Cell lysates and purified ASG samples were denatured and subjected to treatment with peptide N-glycosidase F and endoglucosaminidase H (both from Roche Applied Science) as described (24Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In case of peptide N-glycosidase F deglycosylation, samples were denatured with 0.5% SDS and 0.2% β-mercaptoethanol at pH 7.2 for 5 min at 95 °C. Samples were mixed with peptide N-glycosidase F in incubation buffer containing 1.2% Triton X-100 at pH 7.2. For endoglucosaminidase H treatment, samples were denatured at pH 5.0 with 0.01% SDS and 0.7% β-mercaptoethanol for 5 min at 95 °C and subjected to deglycosylation. In both cases, samples were incubated at 37 °C for 2 or 24 h and analyzed by Western blotting. Western Blot—For Western blot analysis, a mouse monoclonal antibody directed against the RGS-His6 epitope was used as a primary antibody (Qiagen). Signals were detected using a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes) and ECL detection reagent (Pierce). Signals were quantified using the AIDA 4.06 software package (Raytest, Straubenhardt, Germany). Enzymatic Assays—Activities of ASG toward pNCS or p-nitrophenyl sulfate (pNPS) were assayed using 10 mm pNCS or pNPS in either 0.5 m sodium acetate, (pH 4.5–6.0), MES (pH 6.5), or Tris-HCl (pH 7.0–8.0). Samples containing ASG as indicated were incubated at 37 °C for 1 h in 150 μl of reaction volume. Reactions were terminated by addition of 150 μl of 1 m NaOH. Michaelis-Menten kinetic analysis was performed at pH 5.6 using 0.5–30 mm pNCS in 0.5 m sodium acetate. Absorbances were measured at 515 nm (e515 = 12400 m-1 cm-1) in the case of pNCS or at 405 nm (e405 = 18000 m-1 cm-1) for pNPS. Inhibition kinetics included Na2HPO4, NaHSO4, or warfarin (Sigma-Aldrich) at the indicated concentrations. Activity measurements with 4-MUS were performed accordingly, using 10 mm 4-MUS in the respective buffers. Reactions were stopped by addition of 150 μl of 1 m Na2CO3/NaHCO3, pH 10.7. The fluorescence of 4-methylumbelliferone, compared with a calibration curve, was measured with an excitation wavelength of 360 nm and an emission wavelength of 465 nm. All absorbance and fluorescence measurements were performed using an infinite M200 microplate reader (TECAN, Crailsheim, Germany). The temperature optimum was determined using a gradient thermocycler. Immunofluorescence—Stably transfected HT1080 cells expressing ASG-His were grown on poly-l-lysine-coated coverslips for 24 h and labeled with 50 nm LysoTracker Red DND-99 (Molecular Probes) for 2 h in serum-free Dulbecco's modified Eagle's medium. The cells were briefly washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 2% fetal calf serum, the cells were incubated with a mouse monoclonal anti-RGS-His6 antibody (Qiagen) for 1 h. The primary antibody was detected with an Alexa-488-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes). Immunofluorescence images were obtained on a Leica DM5000 B microscope equipped with an HCX PL APO ×100 oil immersion objective. Mannose 6-Phosphate Receptor Binding Assay—10 μg of purified ASG were incubated overnight at 4 °C with an Affi-Gel-10-based affinity matrix (2-ml column volume; Bio-Rad) to which a 1:1 mixture of MPR46/MPR300 purified from goat had been immobilized as described (25Koster A. Saftig P. Matzner U. von Figura K. Peters C. Pohlmann R. EMBO J. 1993; 12: 5219-5223Crossref PubMed Scopus (77) Google Scholar). The column was washed four times with 2 ml of MPR binding buffer (50 mm imidazole, pH 6.5, 150 mm NaCl, 5 mm Na-β-glycerophosphate, 2 mm EDTA, 10 mm MgCl2, 0.2% NaN3) and then three times with 2 ml of MPR binding buffer containing 5 mm glucose 6-phosphate to remove unspecifically bound proteins. Mannose 6-phopshate-containing proteins were eluted with 10 × 1 ml of 5 mm glucose 6-phosphate in MPR binding buffer (26Kollmann K. Mutenda K.E. Balleiniger M. Eckermann E. von Figura K. Schmidt B. Lübke T. Proteomics. 2005; 5: 3966-3978Crossref PubMed Scopus (75) Google Scholar). Wash and eluate fractions were analyzed by Western blotting. Gel Filtration Analysis—Purified ASG was subjected to gel filtration on a Superdex 75 3.2/30 PC column (GE Healthcare), equilibrated with either 50 mm NaAc, 150 mm NaCl, pH 5.6, or 50 mm Tris-HCl, 150 mm NaCl, pH 7.4. Proteins were eluted at a flow rate of 40 μl/min using an ÄKTA Ettan LC system (GE Healthcare). Fractions were examined for ASG activity by pNCS assays. Reverse Transcription PCR Expression Analysis—Reverse transcription PCR experiments were performed using a panel of normalized cDNAs prepared from eight normal human tissues (MTC panel human I; Clontech). An internal 880-bp fragment from human ARSG was amplified by PCR (forward primer 5′-TTCATCCAGCGTGCAAGCACCAGC-3′ and reverse primer 5′-CCTACCAAATTGCCTGCCGCTGTC-3′). PCR was carried out for 35 cycles with 62 °C annealing temperature. Normalization was confirmed by primers specific for glycerol aldehyde-3-phosphate dehydrogenase. Purification and Arylsulfatase Activity of ASG—To investigate the biochemistry and cell biology of ASG, we generated HT1080 cell lines stably expressing human ASG with a C-terminal RGS-His6 tag. The secreted protein was purified from the conditioned medium using a combination of Ni2+ affinity and cation exchange chromatography (Fig. 1, A and B). The purified protein had a size of 63 kDa and was identified as ASG by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides. Purified ASG was obtained in catalytically active form. ASG cleaves the pseudosubstrate pNCS and also, but with lower activity, pNPS and 4-MUS. To find optimal conditions, we analyzed the pH dependence of the reaction with pNCS and 4-MUS and found pH optima at 5.4 and 5.6, respectively (Fig. 1C), thus giving a first indication for a possible lysosomal function of ASG. Furthermore, we analyzed the temperature optimum of ASG (Fig. 1D). During a 60-min incubation, ASG cleaved pNCS most efficiently at 45–50 °C. The corresponding Arrhenius plot was linear over the temperature range from 0 to 30 °C (see supplemental Fig. S1). From this plot, an activation energy of Ea = 43.1 kJ/mol was calculated. ASG activity was retained even at relatively high temperatures above 55 °C, thus characterizing ASG as a thermostable enzyme. The extraordinary stability of ASG is underlined by a half-life of more than 100 days during storage at 4 °C and pH 6.0 (data not shown). Characterization of ASG Mutants/Isoforms—The active site Cα-formylglycine residue is essential for catalysis in all human sulfatases that have been characterized so far (2Diez-Roux G. Ballabio A. Annu. Rev. Genomics Hum. Genet. 2005; 6: 355-379Crossref PubMed Scopus (148) Google Scholar, 13Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The so-called sulfatase signature 84-CSPSR-88 of ASG predicts Cys-84 to be post-translationally modified to FGly. To confirm the importance of FGly in ASG, we mutagenized Cys-84 to Ala and compared the activity of the mutant to the wild-type enzyme by performing pNCS activity assays with cell lysates from stably transfected cells. We also analyzed the activity of the ASG A501P isoform encoded by the sequence of the human expressed sequence tag clone KIAA1001 (see "Experimental Procedures" and Refs. 21Ferrante P. Messali S. Meroni G. Ballabio A. Eur. J. Hum. Genet. 2002; 10: 813-818Crossref PubMed Scopus (47) Google Scholar, 22Nagase T. Ishikawa K. Suyama M. Kikuno R. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1999; 6: 63-70Crossref PubMed Scopus (113) Google Scholar). The ASG C84A mutant showed no activity above background levels resulting from endogenous sulfatases (Fig. 2). Compared with wild-type ASG, the A501P isoform displayed only ∼35% activity. Wild-type ASG was used for all further studies. Arylsulfatase Substrates, Kinetics, and Inhibitors of ASG—To further investigate the enzymology of ASG, we analyzed the influence of substrate concentration on the activity toward pNCS (Fig. 3A). ASG follows Michaelis-Menten kinetics and shows hyperbolic substrate saturation. At substrate concentrations above 15 mm substrate inhibition is observed. The double-reciprocal Lineweaver-Burk transformation (Fig. 3B) yields the following kinetic constants: Km = 4.2 mm, Vmax = 63.5 μmol/(min·mg) = 63.5 units/mg. Compared with other arylsulfatases, these values fall within the normal range. Typical activities toward pNCS are 40–100 units/mg (27Waldow A. Schmidt B. Dierks T. von Bülow R. von Figura K. J. Biol. Chem. 1999; 274: 12284-12288Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Because of its limited solubility at acidic pH, no substrate saturation could be obtained with 4-MUS as a substrate (data not shown). With an estimated Vmax of only 0.2–0.4 units/mg and a Km of 5–15 mm, 4-MUS represents a rather poor substrate for ASG. Compared with pNCS, the cleavage of the closely related pNPS, merely lacking one hydroxyl group, is even slower (∼0.05 units/mg at pH 5.6), indicating a stringent substrate specificity that might also apply for physiological substrates. Many sulfatases are inhibited by their product, sulfate, or by its analog, phosphate (28Chruszcz M. Laidler P. Monkiewicz M. Ortlund E. Lebioda L. Lewinski K. J. Inorg. Biochem. 2003; 96: 386-392Crossref PubMed Scopus (36) Google Scholar). Interestingly, inhibition of ASG by phosphate is much stronger than by sulfate (Fig. 3C). Whereas sulfate has an IC50 value of ∼1 mm, inhibition by phosphate shows an IC50 of ∼50 μm at 10 mm pNCS (Fig. 3C, inset). To determine the type of inhibition, substrate saturation curves were recorded at different phosphate concentrations, which are presented in Fig. 3D as Lineweaver-Burk plots. The regression lines show a common intercept on the ordinate axis as expected for a competitive inhibitor. From the slopes of the regression lines plotted against inhibitor concentrations, a Ki value of 17 μm was extrapolated. In contrast to arylsulfatase E (29Franco B. Meroni G. Parenti G. Levilliers J. Bernard L. Gebbia M. Cox L. Maroteaux P. Sheffield L. Rappold G.A. Andria G. Petit C. Ballabio A. Cell. 1995; 81: 15-25Abstract Full Text PDF PubMed Scopus (259) Google Scholar), ASG is not inhibited by warfarin even at concentrations close to the solubility limit (data not shown). ASG Is Active in the Monomeric State—To analyze whether or not ASG forms dimeric or oligomeric complexes, purified ASG was subjected to gel filtration on a Superdex 75 column at either pH 5.6 or 7.4. Fractions were tested for sulfatase activity (Fig. 4). At both pH values, neither protein UV peaks nor sulfatase activities provided any indication for dimerization or oligomerization of ASG as described for its closest relative, arylsulfatase A (30von Bülow R. Schmidt B. Dierks T. Schwabauer N. Schilling K. Weber E. Usón I. von Figura K. J. Biol. Chem. 2002; 277: 9455-9461Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). ASG eluted as a 63-kDa protein, which corresponds to the monomeric molecular mass also observed in SDS-PAGE. Thus, unlike, for example, arylsulfatase B, ASG does not undergo lysosomal processing (31Steckel F. Hasilik A. von Figura K. J. Biol. Chem. 1983; 258: 14322-14326Abstract Full Text PDF PubMed Google Scholar). Characterization of the ASG Glycosylation State—The ASG sequence contains four potential N-glycosylation sites (asparagine residues 117, 215, 356, and 497). Treatment with peptide N-glycosidase F reduces the apparent sizes of both the intracellular and the secreted protein from a broad, glycosylated band at 63 kDa (with micro-heterogeneity in its N-glycans) to a sharp band of ∼53 kDa (Fig. 5A). This difference in size suggests that all four N-glycosylation sites are utilized, assuming that the average mass is ≤1.9 kDa for a high mannose and ≤2.9 kDa for a complex type oligosaccharide (24Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Furthermore, the intracellular ASG is largely sensitive toward treatment with endoglucosaminidase H, whereas the secreted ASG is resistant (Fig. 5A, lane 8). Thus, high mannose type oligosaccharides added to ASG in the ER are processed to complex type N-glycan structures during maturation in the Golgi. The intracellular ASG represents a mixture containing mainly high mannose N-glycans, one of which, however, was endoglucosaminidase H-resistant in the majority of ASG molecules (Fig. 5A, lane 4). Endoglucosaminidase H treatment did not result in a complete deglycosylation even at extended incubation times and increased endoglucosaminidase H concentrations. ASG Binds to Mannose 6-Phosphate Receptors—Most lysosomal proteins are transported to the lysosomes via the mannose 6-phosphate receptor (M6PR) pathway (32Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (816) Google Scholar, 33Luzio J.P. Pryor P.R. Bright N.A. Nat. Rev. Mol. Cell Biol. 2007; 8: 622-632Crossref PubMed Scopus (1201) Google Scholar). In a comprehensive proteome analysis of lysosomal pr

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