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• W2119272563 abstract "Sialic acid and glucuronic acid are monocarboxylated monosaccharides, which are normally present in sugar side chains of glycoproteins, glycolipids, and glycosaminoglycans. After degradation of these compounds in lysosomes, the free monosaccharides are released from the lysosome by a specific membrane transport system. This transport system is deficient in the human hereditary lysosomal sialic acid storage diseases (Salla disease and infantile sialic acid storage disease, OMIM 269920). The lysosomal sialic acid transporter from rat liver has now been purified to apparent homogeneity in a reconstitutively active form by a combination of hydroxyapatite, lectin, and ion exchange chromatography. A 57-kDa protein correlated with transport activity. The transporter recognized structurally different types of acidic monosaccharides, like sialic acid, glucuronic acid, and iduronic acid. Transport of glucuronic acid was inhibited by a number of aliphatic monocarboxylates (i.e. lactate, pyruvate, and valproate), substituted monocarboxylates, and several dicarboxylates.cis-Inhibition, trans-stimulation, and competitive inhibition experiments with radiolabeled glucuronic acid as well as radiolabeled l-lactate demonstrated thatl-lactate is transported by the lysosomal sialic acid transporter. l-Lactate transport was proton gradient-dependent, saturable with a K mof 0.4 mm, and mediated by a single mechanism. These data show striking biochemical and structural similarities of the lysosomal sialic acid transporter with the known monocarboxylate transporters of the plasma membrane (MCT1, MCT2, MCT3, and Mev). Sialic acid and glucuronic acid are monocarboxylated monosaccharides, which are normally present in sugar side chains of glycoproteins, glycolipids, and glycosaminoglycans. After degradation of these compounds in lysosomes, the free monosaccharides are released from the lysosome by a specific membrane transport system. This transport system is deficient in the human hereditary lysosomal sialic acid storage diseases (Salla disease and infantile sialic acid storage disease, OMIM 269920). The lysosomal sialic acid transporter from rat liver has now been purified to apparent homogeneity in a reconstitutively active form by a combination of hydroxyapatite, lectin, and ion exchange chromatography. A 57-kDa protein correlated with transport activity. The transporter recognized structurally different types of acidic monosaccharides, like sialic acid, glucuronic acid, and iduronic acid. Transport of glucuronic acid was inhibited by a number of aliphatic monocarboxylates (i.e. lactate, pyruvate, and valproate), substituted monocarboxylates, and several dicarboxylates.cis-Inhibition, trans-stimulation, and competitive inhibition experiments with radiolabeled glucuronic acid as well as radiolabeled l-lactate demonstrated thatl-lactate is transported by the lysosomal sialic acid transporter. l-Lactate transport was proton gradient-dependent, saturable with a K mof 0.4 mm, and mediated by a single mechanism. These data show striking biochemical and structural similarities of the lysosomal sialic acid transporter with the known monocarboxylate transporters of the plasma membrane (MCT1, MCT2, MCT3, and Mev). N-acetylneuraminic acid glucuronic acid 2-(N-morpholino)-ethanesulfonic acid iduronic acid 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid polyacrylamide gel electrophoresis. The major function of lysosomes is the degradation of a large variety of intra- and extracellular macromolecules. The release of degradation products from the lysosome is accomplished by specific membrane transport systems. More than 20 lysosomal transporters have been characterized for specific solutes like amino acids, sugars, nucleosides, ions, and vitamins (1Pisoni R.L. Thoene J.G. Biochim. Biophys. Acta. 1991; 1071: 351-373Crossref PubMed Scopus (94) Google Scholar). Their fundamental role in biology is illustrated by the occurrence of two human inherited diseases with a defective lysosomal transport function, cystinosis and sialic acid storage diseases (2Gahl W.A. Schneider J.A. Aula P.P. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 3763-3797Google Scholar). Sialic acid storage diseases are autosomal recessive disorders that are characterized by mental retardation and a variable degree of neurodegeneration. Lysosomal accumulation and excessive urinary excretion of free sialic acid are pathognomonic findings. Previously, we have characterized a carrier in the lysosomal membrane with substrate specificity for the acidic monosaccharides sialic acid (Neu5Ac)1, uronic acids, and aldonic acids (3Mancini G.M.S. de Jonge H.R. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: 15247-15254Abstract Full Text PDF PubMed Google Scholar). Subsequent studies in our laboratory showed that a defective transport of sialic and glucuronic acid (GlcA) is the primary defect in both clinical variants (4Mancini G.M.S. Beerens C.E.M.T. Aula P.P. Verheijen F.W. J. Clin. Invest. 1991; 87: 1329-1335Crossref PubMed Scopus (63) Google Scholar), Salla disease and infantile sialic acid storage disease. Recently, the gene for these disorders has been localized to the same refined chromosomal area on 6q14-q15 by linkage disequilibrium analysis (5Schleutker J. Leppänen P. Månsson J.E. Erikson A. Weissenbach J. Peltonen L. Aula P. Am. J. Hum. Genet. 1995; 57: 893-901PubMed Google Scholar). However, the disease gene has not been identified yet. The elucidation of the molecular structure and functional properties of the lysosomal sialic acid transporter is indispensable for further understanding of the molecular defect(s) in the clinical heterogeneous forms of sialic acid storage diseases. Previously, we have developed a functional reconstitution system for the sialic acid transporter that provided the tool to start the purification and functional characterization of the transport protein (6Mancini G.M.S. Beerens C.E.M.T. Galjaard H. Verheijen F.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6609-6613Crossref PubMed Scopus (24) Google Scholar). In this paper we present the purification of the sialic acid transporter from lysosomal membranes of rat liver to apparent homogeneity. Its functional properties are compared with those of other monocarboxylate transporters present in the plasma membrane of various mammalian cells (7Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Crossref PubMed Google Scholar, 8Garcia C.K. Goldstein J.L. Pathak R.K. Anderson R.G.W. Brown M.S. Cell. 1994; 76: 865-873Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 9Garcia C.K. Brown M.S. Pathak R.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 1843-1849Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Highly purified lysosomal membrane vesicles were isolated from livers of adult Wistar rats (3Mancini G.M.S. de Jonge H.R. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: 15247-15254Abstract Full Text PDF PubMed Google Scholar). The lysosomal membrane vesicles were suspended at a protein concentration of 8–10 mg/ml in 20 mm NaHepes, pH 7.4, 1 mm EDTA and were stored at −70 °C. All chemicals used were obtained from Sigma or as indicated. l-Iduronic acid, sodium salt was obtained from Toronto Research Chemicals Inc. (North York, ON, Canada). All the tested carboxylates were titrated with NaOH before use. Reconstitution of the protein eluates into liposomes was performed as described earlier (6Mancini G.M.S. Beerens C.E.M.T. Galjaard H. Verheijen F.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6609-6613Crossref PubMed Scopus (24) Google Scholar), with the following modification: proteoliposomes were formed by incubating the protein sample, containing detergent and phospholipid (total volume, 170 μl), with 150 μl of Amberlite XAD-2 beads (Fluka) in 20 mm NaHepes, pH 7.4, 100 mm KCl. After 30 min of rotation at room temperature, beads were removed by short centrifugation, and proteoliposomes were used for transport assays. After reconstitution, the carrier activity was assayed by uptake of radiolabeled GlcA in the presence of an inwardly directed proton gradient. Because Neu5Ac and GlcA are transported by the same lysosomal transporter for acidic monosaccharides (3Mancini G.M.S. de Jonge H.R. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: 15247-15254Abstract Full Text PDF PubMed Google Scholar, 4Mancini G.M.S. Beerens C.E.M.T. Aula P.P. Verheijen F.W. J. Clin. Invest. 1991; 87: 1329-1335Crossref PubMed Scopus (63) Google Scholar), we have performed all studies using radiolabeled GlcA, which was more readily available. Aliquots of proteoliposomes (25 μl) were incubated at 37 °C with 5 μl of 240 mm Mes (free acid) containing 2 μCi ofd-[1-3H]GlcA (Amersham Pharmacia Biotech; specific activity, 6.6 Ci/mmol), resulting in an extravesicular pH of 5.5 and a final concentration of 10 μm GlcA. Blank values were determined by incubation of proteoliposomes at 37 °C with 40 mm Mes (free acid), 7 mm unlabeled NaGlcA, pH 5.5, and 2 μCi of d-[1-3H]GlcA and subtracted from all determinations. Previous experiments showed that uptake rates are linear up to 1 min. After 1 min, the reactions were stopped by diluting the sample with 70 μl of ice-cold incubation buffer (17 mm NaHepes, 84 mm KCl, 40 mm Mes (free acid), pH 5.5). The samples were immediately applied to a Sephadex G50 fine (Amersham Pharmacia Biotech) column (Pasteur pipettes, 0.5 × 5cm) at 4 °C. Columns were equilibrated in cold incubation buffer, and vesicles were eluted with 1 ml of cold incubation buffer. Vesicle-associated radioactivity was determined by liquid scintillation counting in 10 ml of Insta-gel (Packard). cis-Inhibition experiments were performed by incubating the proteoliposomes for 1 min at 37 °C with 2 μCi of [3H]GlcA (final concentration, 10 μm) in 40 mm Mes (free acid), resulting in an inwardly directed proton gradient (pHin = 7.4 > pHout = 5.5), and 7 mm of the tested compound. For trans-stimulation studies, a 60% proteoliposome solution (25 μl) was pre-incubated for 60 min at 37 °C with 17 mm NaHepes, 84 mm KCl, 40 mm Mes acid, pH 5.5, plus 10 μm monensin, 10 μmvalinomycin (Boehringer Mannheim) in the presence or absence of 1 mm unlabeled substrate. The assay was started by adding 75 μl of an equivalent buffer containing 2 μCi of [3H]GlcA at 37 °C with a final concentration of 0.25 mm. When the samples were pre-incubated without unlabeled substrate, the external final concentration was corrected as in the case of preloading (0.25 mm unlabeled compound). After 1 min, the reaction was stopped as described (6Mancini G.M.S. Beerens C.E.M.T. Galjaard H. Verheijen F.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6609-6613Crossref PubMed Scopus (24) Google Scholar). The experiments with [14C]l-lactate (Amersham Pharmacia Biotech; specific activity, 152 mCi/mmol) were largely performed as described for [3H]GlcA. However, incubation mixtures contained 0.066 μCi of [14C]l-lactate (final concentration, 15 μm) and were performed at 20 °C instead of 37 °C. Blank values were determined by incubation of proteoliposomes with 40 mm Mes (free acid), 7 mm unlabeled sodiuml-lactate and subtracted from all determinations. For protein side chain modification, proteoliposomes (100 μl) were incubated and treated as described (6Mancini G.M.S. Beerens C.E.M.T. Galjaard H. Verheijen F.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6609-6613Crossref PubMed Scopus (24) Google Scholar). For a single purification, lysosomal membrane vesicles prepared from 150 g of rat livers (15 rats) were used. Solubilization of lysosomal membrane proteins was performed by mixing the lysosomal membrane vesicles 1:1 (v/v) with 1% Triton X-100 (especially purified for membrane research, Boehringer Mannheim), 20 mm Tris-HCl, pH 7.4. After 25 min of incubation at 0 °C, unextracted material was pelleted by ultracentrifugation at 150,000 × g in a Beckman SW 40 rotor for 20 min at 4 °C. The Triton X-100 extract was applied to hydroxyapatite columns (Pasteur pipettes containing 0.5 g of dry material, Biogel HTP, Bio-Rad, packed by 15 s of tapping) at 4 °C, with a maximum of 500 μl solubilized material/column. Each column was washed with 3 ml of 20 mm Tris-HCl, pH 7.4, 0.1% Triton X-100 (buffer A). Elution was with 3 ml of buffer A, 25 mm Na2HPO4, NaH2PO4, pH 7.4. After pooling all the 3-ml eluates, a 2-ml sample was concentrated in a Centricon 10 device (Amicon, Inc., Beverly, MA) until 100–150 μl and desalted. A 50-μl aliquot was used for the reconstitution assay and a 20-μl aliquot was used for the protein assay. Desalting was performed on a 2-ml Sephadex G50 medium (Amersham Pharmacia Biotech) column equilibrated in buffer A (10Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar). The eluates of the different hydroxyapatite columns were pooled and applied to a 2-ml lentil lectin affinity chromatography column (lentil lectin-Sepharose 4B, Amersham Pharmacia Biotech) pre-equilibrated in buffer A. After washing the lentil lectin column with 2 ml of buffer A, the flow-through fraction (unretained material) was applied to a 2-ml DEAE-Sephacel (Amersham Pharmacia Biotech) anion exchanger pre-equilibrated in buffer A containing 10% glycerol (buffer B). A 2-ml sample of the lentil lectin flow-through fraction was concentrated in a Centricon 10 device to 100–150 μl and desalted (10Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar). A 50-μl aliquot was used for the reconstitution assay, and a 20-μl aliquot was used for the protein assay. After extensive washing the DEAE-Sephacel column with 20 ml of buffer B and 20 ml of buffer B with 40 mm NaCl, bound material was eluted with 6 ml of buffer B with 100 mm NaCl. This fraction was stored at −70 °C. After the DEAE-eluate was thawed, a 0.5 ml sample was concentrated in a Centricon 10 device until 100 μl and desalted (10Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar). A 50 μl aliquot was used for the reconstitution assay and a 20 μl aliquot for the protein assay. The 100 mm NaCl DEAE-eluate (5.5 ml) was adjusted to pH 6.0 with 0.5 m Mes (free acid) and applied to a prepacked hydroxyapatite column (1-ml EconoPac HTP cartridge, Bio-Rad) pre-equilibrated in 300 mm NaCl, 20 mm NaMes pH 6.0, 0.1% Triton X-100. Transport activity was eluted with 6 ml of 1 mm Na2HPO4, NaH2PO4, pH 6.0, 300 mm NaCl, 0.1% Triton X-100. This eluate was concentrated in Millipore ultrafree-15 centrifugal filters 10K (Millipore Corporation, Bedford) to approximately 300 μl and desalted (10Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar). A 50-μl aliquot was used for the reconstitution assay, and a 100-μl aliquot was used for the protein assay. The concentrated hydroxyapatite eluate (150 μl) was applied to a 0.10-ml Mono Q anion exchange column attached to a Amersham Pharmacia Biotech SMART system. This column was equilibrated in buffer B, and bound material was eluted with a linear gradient of 0–210 mm NaCl in buffer B. Fractions of 0.1 ml were collected and pooled pairwise, buffer was exchanged for 20 mm NaHepes, 100 mm KCl, 0.1% Triton X-100 by the desalting procedure as described above, and a 50-μl aliquot was used for the reconstitution assay. All column procedures were performed at 4 °C. The purity of the various active fractions was determined by SDS-polyacrylamide gel electrophoresis according to Laemmli (11Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) of methanol/chloroform precipitated samples (12Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar), followed by Coomassie Brilliant Blue R-250 or the silver nitrate staining according to Amersham Pharmacia Biotech. Protein concentration was determined by the procedure of Lowry et al. as modified by Peterson (13Peterson G.L. Anal. Biochem. 1979; 100: 201-220Crossref PubMed Scopus (883) Google Scholar) for the presence of Triton. Protein concentrations in eluates of the second hydroxyapatite column were determined after methanol/chloroform precipitation (12Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar). Protein concentrations in Mono Q eluates were too low to be determined by the above assay and were therefore estimated from silver-stained SDS-PAGE gels. For the endoglycosidase F/N-Glycosidase F (Boehringer Mannheim) treatment of the purified protein, the Mono Q fractions 19–23 were pooled, concentrated, and incubated with 25 milliunits endoglycosidase F, 100 μl in the presence of 20 mmpotassium phosphate buffer, pH 7.4, 50 mm EDTA, 2% Triton X-100, 0.2% SDS, 2% β-mercaptoethanol for 2 h at 37 °C. Proteins were precipitated with methanol/chloroform (12Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar). The pellet was resuspended in sample buffer and analyzed by SDS-PAGE (10% gel). Various membrane (transport) proteins have been successfully purified using hydroxyapatite as well as ion exchange chromatography in the presence of detergents (14Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 15Kyouden T. Himeno M. Ishikawa T. Ohsumi Y. Kato K. J. Biochem. (Tokyo). 1992; 111: 770-777Crossref PubMed Scopus (12) Google Scholar, 16Arai K. Shimaya A. Hiratani N. Ohkuma S. J. Biol. Chem. 1993; 268: 5649-5660Abstract Full Text PDF PubMed Google Scholar, 17Kim S. Ezaki J. Himeno M. Kato K. J. Biochem. (Tokyo). 1993; 114: 126-131Crossref PubMed Scopus (21) Google Scholar, 18Schulte S. Stoffel W. Eur. J. Biochem. 1995; 233: 947-953Crossref PubMed Scopus (20) Google Scholar). In addition, affinity chromatography with oligosaccharide-specific lectins has been used to identify the major heavily glycosylated lysosomal membrane proteins: LAMPs (lysosomal-associated membraneproteins) and LIMPs (lysosomalintegral membrane proteins) (19Carlsson S.R. Roth J. Piller F. Fukuda M. J. Biol. Chem. 1988; 263: 18911-18919Abstract Full Text PDF PubMed Google Scholar, 20Akasaki K. Yamaguchi Y. Furuno K. Tsuji H. J. Biochem. (Tokyo). 1991; 110: 922-927Crossref PubMed Scopus (17) Google Scholar, 21Okazaki I. Himeno M. Ezaki J. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1992; 111: 763-769Crossref PubMed Scopus (36) Google Scholar, 22Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar). Based on the success of these purification methods for membrane proteins we developed a purification protocol for the lysosomal sialic acid transporter. Previously, we have reported a successful reconstitution procedure for the rat liver lysosomal sialic acid transporter that now provided the functional assay to follow fractionation and purification of the solubilized transporter (6Mancini G.M.S. Beerens C.E.M.T. Galjaard H. Verheijen F.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6609-6613Crossref PubMed Scopus (24) Google Scholar). At all steps of the purification procedure, samples were collected and reconstituted into proteoliposomes, and their transport activities were measured using radiolabeled GlcA as a substrate (TableI).Table IPurification of the sialic acid transporter from rat liver lysosomal membrane vesiclesFractionProteinTotal proteinTotal ActivityYieldSpecific activityFold enhancementμg/mlμgpmol/min%pmol GlcA/mg/minSolubilized lysosomal membrane extract200120002102.4100175.21First hydroxyapatite eluate16.4591441.521747.34Lentil lectin eluate13.6489378.418774.44.4DEAE eluate16.699.5252.3122534.914.5Second hydroxyapatite eluate0.53.042.1214171.480Mono Q eluate0.140.0282.10.175757.6432Lysosomal membrane vesicles (approximately 25 mg of protein) derived from 150 g of rat livers were used as starting material. The purification procedure, reconstitution, and transport assay were performed as described under “Experimental Procedures.” Activity is expressed as uptake of [3H]GlcA in 1 min at 37 °C. Data represent the means of three separate isolations. Open table in a new tab Lysosomal membrane vesicles (approximately 25 mg of protein) derived from 150 g of rat livers were used as starting material. The purification procedure, reconstitution, and transport assay were performed as described under “Experimental Procedures.” Activity is expressed as uptake of [3H]GlcA in 1 min at 37 °C. Data represent the means of three separate isolations. The Triton X-100 solubilized lysosomal membrane proteins were applied to small columns of dry hydroxyapatite material. The columns were washed with equilibration buffer at pH 7.4, and about 20% of the transport activity was eluted with 25 mm sodium phosphate buffer at pH 7.4. This resulted in a 4-fold purification. The next step consisted of lentil lectin affinity chromatography. Almost all activity of the sialic acid transporter was recovered from the column flow-through. Lentil lectin recognizes α-d-glucose and α-d-mannose residues and therefore binds glycoproteins. Consequently, a number of major lysosomal membrane glycoproteins bound to the column and thus could be separated from the protein preparation containing transport activity. This step was kept in our protocol despite the fact that it did not lead to an increase in specific activity. The lentil lectin flow-through fraction was applied to a DEAE-Sephacel anion exchange column. With 100 mm NaCl, 12% of the total transport activity was eluted. As depicted in Table I, this resulted in a ≈14.5-fold increase in specific activity over the starting material. Analysis of the protein composition of fractions obtained from these initial purification steps is shown in Fig.1. Many different protein bands were still present. The next purification step consisted of chromatography on hydroxyapatite. This time the column was pre-equilibrated at pH 6.0 in the presence of 300 mm NaCl. Under these conditions, acidic proteins are retained and are eluted with low phosphate buffers. This step provided an important purification of the sialic acid transport protein with an 80-fold enrichment in specific activity (Table I). SDS-PAGE protein analysis using silver staining showed at least four distinct protein bands (Fig.2 A). One of these proteins has a molecular mass of 85 kDa and based on its N-terminal amino acid sequence represented one of the major lysosomal membrane glycoproteins, the Lgp85 or LIMP II (23Fujita H. Ezaki J. Noguchi Y. Kono A. Himeno M. Kato K. Biochem. Biophys. Res. Commun. 1991; 178: 444-452Crossref PubMed Scopus (32) Google Scholar). Another major 67-kDa protein represented the lysosomal membrane-bound subunit of acid phosphatase (24Himeno M. Koutoku H. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1989; 105: 449-456Crossref PubMed Scopus (30) Google Scholar). The other proteins were considered as candidates for the lysosomal sialic acid transporter. The next purification step consisted of a strong anion exchange Mono Q column attached to the SMART system of Amersham Pharmacia Biotech. Retained proteins were eluted with a gradient of 0–210 mmNaCl. SDS-PAGE analysis by silver nitrate staining of the eluted proteins showed a predominant protein band with a molecular mass of ≈57 kDa in the fractions 20/21 in which also the highest GlcA transport activity was observed (Fig. 2 B). In addition, quantitative image analysis of the SDS-PAGE protein elution pattern from the Mono Q column demonstrated a correlation between the 57-kDa protein and the transport activity (data not shown). All other visualized proteins could not represent the sialic acid transporter, because they became more prevalent in following fractions, where lower or no transport activity was detected (Fig. 2 B). In the final protein preparations (fractions 20–21 from the Mono Q column) transport activity was 432-fold enriched over the activity in the initial lysosomal membrane extract (Table I). Considering that the lysosomal membrane marker β-glucosidase is about 100-fold enriched in the lysosomal membrane vesicles (used as a starting material), the sialic acid transport protein is about 40,000-fold purified in the final eluate of the Mono Q column. To investigate the glycosylation of the transporter, the final protein preparation was incubated with the enzyme mixture endoglycosidase F/N-glycosidase F. After treatment, the apparent molecular mass of the 57-kDa protein was not decreased. The apparent molecular mass of a control glycoprotein was decreased as a result of cleavage of glycosydic chains (data not shown). This, together with the observation that this protein did not interact with lentil lectin, indicates that the carrier is apparently not glycosylated. Analysis by SDS-PAGE in the presence or absence of the thiol-reducing agent 2-mercaptoethanol did not show any alteration of the electrophoretic behavior of the purified transport protein (data not shown). This indicates that the transporter is not functional as a (homo)dimer or polymer linked by disulfide bridges. Because the final yield of the highly purified sialic acid transporter was very low, detailed kinetic studies were difficult to perform. Therefore, most kinetic characterization of the lysosomal sialic acid transporter was performed using partially purified preparations (DEAE-Sephacel eluates). Subsequently, some key experiments were repeated in a concise manner with the highly purified transport preparation. In earlier substrate specificity studies with the crude lysosomal sialic acid transporter, we have shown that this transporter recognizes structurally different types of acidic monosaccharides (i.e. the sialic acid Neu5Ac and the uronic acid GlcA) (3Mancini G.M.S. de Jonge H.R. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: 15247-15254Abstract Full Text PDF PubMed Google Scholar,4Mancini G.M.S. Beerens C.E.M.T. Aula P.P. Verheijen F.W. J. Clin. Invest. 1991; 87: 1329-1335Crossref PubMed Scopus (63) Google Scholar). The uronic acid iduronate (IdoA) represents, like GlcA, a major component of glycosaminoglycans. These are degraded in lysosomes, and thus free IdoA is like GlcA expected to be transported across the lysosomal membrane. The recent commercial availability of free IdoA made it now possible to investigate by cis-inhibition andtrans-stimulation studies whether this uronic acid is also a substrate for the lysosomal sialic acid transporter (TableII). IdoA inhibited [3H]GlcA uptake, although less efficiently than Neu5Ac and GlcA. Furthermore, IdoA was able to induce, like its isomer GlcA, almost a 2-fold trans-stimulation (Table II). These experiments indicate that IdoA is indeed a substrate for the sialic acid transporter.Table IIcis-Inhibition and trans-stimulation of [3H]GlcA uptake by mono-, di-, or tricarboxylic acidsTransport activitytrans-Stimulationpmol/mg/min% of control% of not trans-stimulatedControl2280.5 ± 132.6Acidic monosaccharidesGlcA00200Neu5Ac00IdoA916.1 ± 272.440180MonocarboxylatesOxamate553.0 ± 45.524Pyruvate534.6 ± 46.024l-Lactate195.6 ± 67.881504-OH-butyrate369.4 ± 107.216Mevalonate510.0 ± 24.022109Valproate00DicarboxylatesSuccinate0079Malate366.0 ± 21.816Malonate685.3 ± 37.530Maleate592.5 ± 79.526Fumarate234.3 ± 81.010α-Ketoglutarate1090.2 ± 199.648Glutamate2437.8 ± 195.4107TricarboxylateCitrate1665.9 ± 30.673The partially purified (DEAE-Sephacel eluate) sialic acid transporter was reconstituted, and proteoliposomes were incubated 1 min at 37 °C with 10 μm [3H]GlcA in the presence of an inwardly directed proton gradient and 7 mm of the indicated compounds. Data represent the means of four independent determinations ± S.D. In trans-stimulation experiments partially purified proteoliposomes were preincubated for 60 min at 37 °C in the presence or absence of 1 mm unlabeled GlcA, IdoA, l-lactate, mevalonate, or succinate in 20 mm NaHepes, 100 mm KCl, 40 mm Mes, pH 5.5, 10 μm valinomycin and monensin. The transport assay was started by a 4-fold dilution in pH 5.5 incubation buffer with 2 μCi of [3H]GlcA and allowed to proceed for 1 min. In the samples that were preincubated without unlabeled compound, 0.25 mm unlabeled compound was added together with radiolabeled substrate to give the same extravesicular substrate concentration in both experiments. Open table in a new tab The partially purified (DEAE-Sephacel eluate) sialic acid transporter was reconstituted, and proteoliposomes were incubated 1 min at 37 °C with 10 μm [3H]GlcA in the presence of an inwardly directed proton gradient and 7 mm of the indicated compounds. Data represent the means of four independent determinations ± S.D. In trans-stimulation experiments partially purified proteoliposomes were preincubated for 60 min at 37 °C in the presence or absence of 1 mm unlabeled GlcA, IdoA, l-lactate, mevalonate, or succinate in 20 mm NaHepes, 100 mm KCl, 40 mm Mes, pH 5.5, 10 μm valinomycin and monensin. The transport assay was started by a 4-fold dilution in pH 5.5 incubation buffer with 2 μCi of [3H]GlcA and allowed to proceed for 1 min. In the samples that were preincubated without unlabeled compound, 0.25 mm unlabeled compound was added together with radiolabeled substrate to give the same extravesicular substrate concentration in both experiments. We investigated the interaction of the transport protein with other known substrates for organic anion carriers. Initially, mono-, di-, and tricarboxylic acids were tested for theircis-inhibition effect on the initial linear rate of proton-driven [3H]GlcA uptake in a partially purified preparation (Table II). Most of these organic anions are known substrates for the proton-driven monocarboxylate transporters MCT1, MCT2, and MCT3 of the plasma membrane and for the pyruvate and t" @default.
• W2119272563 created "2016-06-24" @default.
• W2119272563 creator A5005464442 @default.
• W2119272563 creator A5008883491 @default.
• W2119272563 creator A5031202008 @default.
• W2119272563 creator A5034736367 @default.
• W2119272563 creator A5064881055 @default.
• W2119272563 date "1998-12-01" @default.
• W2119272563 modified "2023-09-30" @default.
• W2119272563 title "Purification of the Lysosomal Sialic Acid Transporter" @default.
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