THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 273, No. 51, Issue of December 18, pp. 34568–34574, 1998
`Printed in U.S.A.
`
`Purification of the Lysosomal Sialic Acid Transporter
`FUNCTIONAL CHARACTERISTICS OF A MONOCARBOXYLATE TRANSPORTER*
`
`(Received for publication, March 31, 1998, and in revised form, September 23, 1998)
`
`Adrie C. Havelaar, Grazia M. S. Mancini, Cecile E. M. T. Beerens, Ragonda M. A. Souren,
`and Frans W. Verheijen‡
`From the Department of Clinical Genetics, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands
`
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`monosaccharides sialic acid (Neu5Ac)1, uronic acids, and al-
`donic acids (3). Subsequent studies in our laboratory showed
`that a defective transport of sialic and glucuronic acid (GlcA) is
`the primary defect in both clinical variants (4), Salla disease
`and infantile sialic acid storage disease. Recently, the gene for
`these disorders has been localized to the same refined chromo-
`somal area on 6q14-q15 by linkage disequilibrium analysis (5).
`However, the disease gene has not been identified yet. The
`elucidation of the molecular structure and functional proper-
`ties 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 puri-
`fication and functional characterization of the transport
`protein (6).
`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 (7–9).
`
`EXPERIMENTAL PROCEDURES
`Materials
`Highly purified lysosomal membrane vesicles were isolated from
`livers of adult Wistar rats (3). 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 270 °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, Can-
`ada). All the tested carboxylates were titrated with NaOH before use.
`
`Reconstitution
`Reconstitution of the protein eluates into liposomes was performed
`as described earlier (6), with the following modification: proteolipo-
`somes were formed by incubating the protein sample, containing deter-
`gent and phospholipid (total volume, 170 ml), with 150 ml 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.
`
`Transport Assay
`After reconstitution, the carrier activity was assayed by uptake of
`radiolabeled GlcA in the presence of an inwardly directed proton gra-
`dient. Because Neu5Ac and GlcA are transported by the same lysosomal
`transporter for acidic monosaccharides (3, 4), we have performed all
`studies using radiolabeled GlcA, which was more readily available.
`Aliquots of proteoliposomes (25 ml) were incubated at 37 °C with 5 ml of
`240 mM Mes (free acid) containing 2 mCi of D-[1-3H]GlcA (Amersham
`Pharmacia Biotech; specific activity, 6.6 Ci/mmol), resulting in an ex-
`travesicular pH of 5.5 and a final concentration of 10 mM GlcA. Blank
`
`1 The abbreviations used are: Neu5Ac, N-acetylneuraminic acid;
`GlcA, glucuronic acid; Mes, 2-(N-morpholino)-ethanesulfonic acid; IdoA,
`iduronic acid; DIDS, 4,49-diisothiocyanostilbene-2,29-disulfonic acid;
`PAGE, polyacrylamide gel electrophoresis.
`34568
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`This paper is available on line at http://www.jbc.org
`
`Sialic acid and glucuronic acid are monocarboxylated
`monosaccharides, which are normally present in sugar
`side chains of glycoproteins, glycolipids, and glycosami-
`noglycans. After degradation of these compounds in ly-
`sosomes, the free monosaccharides are released from
`the lysosome by a specific membrane transport system.
`This transport system is deficient in the human heredi-
`tary 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 recon-
`stitutively active form by a combination of hydroxyap-
`atite, 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 monocarboxy-
`lates (i.e. lactate, pyruvate, and valproate), substituted
`monocarboxylates, and several dicarboxylates. cis-Inhi-
`bition, trans-stimulation, and competitive inhibition ex-
`periments with radiolabeled glucuronic acid as well as
`radiolabeled L-lactate demonstrated that L-lactate is
`transported by the lysosomal sialic acid transporter.
`L-Lactate transport was proton gradient-dependent, sat-
`urable with a Km of 0.4 mM, and mediated by a single
`mechanism. These data show striking biochemical and
`structural similarities of the lysosomal sialic acid trans-
`porter with the known monocarboxylate transporters of
`the plasma membrane (MCT1, MCT2, MCT3, and Mev).
`
`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 (1). Their
`fundamental role in biology is illustrated by the occurrence of
`two human inherited diseases with a defective lysosomal trans-
`port function, cystinosis and sialic acid storage diseases (2).
`Sialic acid storage diseases are autosomal recessive disorders
`that are characterized by mental retardation and a variable
`degree of neurodegeneration. Lysosomal accumulation and ex-
`cessive 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
`
`* This work was supported in part by the Dutch Organization for
`Scientific Research (NWO). The costs of publication of this article were
`defrayed in part by the payment of page charges. This article must
`therefore be hereby marked “advertisement” in accordance with 18
`U.S.C. Section 1734 solely to indicate this fact.
`‡ To whom correspondence should be addressed. Tel.: 31-10-4087350;
`Fax: 31-10-4362536; E-mail: verheijen@ikg.fgg.eur.nl.
`
`Ranbaxy Ex. 1039
`IPR Petition - USP 9,050,302
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`Purification of the Lysosomal Sialic Acid Transporter
`
`34569
`
`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 mCi of
`D-[1-3H]GlcA and subtracted from all determinations. Previous experi-
`ments showed that uptake rates are linear up to 1 min. After 1 min, the
`reactions were stopped by diluting the sample with 70 ml 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 3 5cm)
`at 4 °C. Columns were equilibrated in cold incubation buffer, and ves-
`icles 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 proteo-
`liposomes for 1 min at 37 °C with 2 mCi of [3H]GlcA (final concentration,
`10 mM) in 40 mM Mes (free acid), resulting in an inwardly directed
`proton gradient (pHin 5 7.4 . pHout 5 5.5), and 7 mM of the tested
`compound.
`For trans-stimulation studies, a 60% proteoliposome solution (25 ml)
`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 mM monensin, 10 mM valinomycin
`(Boehringer Mannheim) in the presence or absence of 1 mM unlabeled
`substrate. The assay was started by adding 75 ml of an equivalent buffer
`containing 2 mCi of [3H]GlcA at 37 °C with a final concentration of 0.25
`mM. When the samples were pre-incubated without unlabeled sub-
`strate, 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 (6).
`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 mCi of [14C]L-
`lactate (final concentration, 15 mM) and were performed at 20 °C instead
`of 37 °C. Blank values were determined by incubation of proteolipo-
`somes with 40 mM Mes (free acid), 7 mM unlabeled sodium L-lactate and
`subtracted from all determinations. For protein side chain modification,
`proteoliposomes (100 ml) were incubated and treated as described (6).
`
`Purification of the Rat Liver Lysosomal Membrane
`Sialic Acid Transport Protein
`For a single purification, lysosomal membrane vesicles prepared
`from 150 g of rat livers (15 rats) were used.
`Step 1: Solubilization—Solubilization of lysosomal membrane pro-
`teins 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 incu-
`bation at 0 °C, unextracted material was pelleted by ultracentrifugation
`at 150,000 3 g in a Beckman SW 40 rotor for 20 min at 4 °C.
`Step 2: Hydroxyapatite—The Triton X-100 extract was applied to
`hydroxyapatite columns (Pasteur pipettes containing 0.5 g of dry ma-
`terial, Biogel HTP, Bio-Rad, packed by 15 s of tapping) at 4 °C, with a
`maximum of 500 ml 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 ml
`and desalted. A 50-ml aliquot was used for the reconstitution assay and
`a 20-ml aliquot was used for the protein assay. Desalting was performed
`on a 2-ml Sephadex G50 medium (Amersham Pharmacia Biotech) col-
`umn equilibrated in buffer A (10).
`Step 3: Lentil Lectin—The eluates of the different hydroxyapatite
`columns were pooled and applied to a 2-ml lentil lectin affinity chro-
`matography 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 ml and
`desalted (10). A 50-ml aliquot was used for the reconstitution assay, and
`a 20-ml aliquot was used for the protein assay.
`Step 4: DEAE-Sephacel—After extensive washing the DEAE-Sepha-
`cel 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 270 °C.
`Step 5: Hydroxyapatite—After the DEAE-eluate was thawed, a 0.5
`ml sample was concentrated in a Centricon 10 device until 100 ml and
`desalted (10). A 50 ml aliquot was used for the reconstitution assay and
`a 20 ml 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 ml and desalted
`(10). A 50-ml aliquot was used for the reconstitution assay, and a 100-ml
`aliquot was used for the protein assay.
`Step 6: Mono Q—The concentrated hydroxyapatite eluate (150 ml)
`was applied to a 0.10-ml Mono Q anion exchange column attached to a
`Amersham Pharmacia Biotech SMART system. This column was equil-
`ibrated 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 Na-
`Hepes, 100 mM KCl, 0.1% Triton X-100 by the desalting procedure as
`described above, and a 50-ml aliquot was used for the reconstitution
`assay. All column procedures were performed at 4 °C.
`
`Protein Characterization and Determination
`The purity of the various active fractions was determined by SDS-
`polyacrylamide gel electrophoresis according to Laemmli (11) of meth-
`anol/chloroform precipitated samples (12), followed by Coomassie Bril-
`liant Blue R-250 or the silver nitrate staining according to Amersham
`Pharmacia Biotech. Protein concentration was determined by the pro-
`cedure of Lowry et al. as modified by Peterson (13) for the presence of
`Triton. Protein concentrations in eluates of the second hydroxyapatite
`column were determined after methanol/chloroform precipitation (12).
`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 ml in the presence of 20 mM potassium phosphate buffer, pH 7.4,
`50 mM EDTA, 2% Triton X-100, 0.2% SDS, 2% b-mercaptoethanol for
`2 h at 37 °C. Proteins were precipitated with methanol/chloroform (12).
`The pellet was resuspended in sample buffer and analyzed by SDS-
`PAGE (10% gel).
`
`RESULTS
`Purification of the Lysosomal Sialic Acid Transporter—Var-
`ious membrane (transport) proteins have been successfully pu-
`rified using hydroxyapatite as well as ion exchange chromatog-
`raphy in the presence of detergents (14–18). In addition,
`affinity chromatography with oligosaccharide-specific lectins
`has been used to identify the major heavily glycosylated lyso-
`somal membrane proteins: LAMPs (lysosomal-associated mem-
`brane proteins) and LIMPs (lysosomal integral membrane pro-
`teins) (19–22). 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 (6). At all steps of the purification
`procedure, samples were collected and reconstituted into pro-
`teoliposomes, and their transport activities were measured us-
`ing radiolabeled GlcA as a substrate (Table I).
`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 trans-
`porter was recovered from the column flow-through. Lentil
`lectin recognizes a-D-glucose and a-D-mannose residues and
`therefore binds glycoproteins. Consequently, a number of ma-
`jor lysosomal membrane glycoproteins bound to the column
`and thus could be separated from the protein preparation con-
`taining transport activity. This step was kept in our protocol
`
`

`
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`Purification of the Lysosomal Sialic Acid Transporter
`
`TABLE I
`Purification of the sialic acid transporter from rat liver lysosomal membrane vesicles
`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.
`
`Fraction
`
`Protein
`
`Total protein
`
`Total Activity
`
`Yield
`
`Specific activity
`
`Solubilized lysosomal
`membrane extract
`First hydroxyapatite eluate
`Lentil lectin eluate
`DEAE eluate
`Second hydroxyapatite
`eluate
`Mono Q eluate
`
`mg/ml
`200
`
`16.4
`13.6
`16.6
`0.5
`
`0.14
`
`mg
`12000
`
`591
`489
`99.5
`3.0
`
`0.028
`
`pmol/min
`2102.4
`
`441.5
`378.4
`252.3
`42.1
`
`2.1
`
`%
`100
`
`21
`18
`12
`2
`
`0.1
`
`pmol GlcA/mg/min
`175.2
`
`747.3
`774.4
`2534.9
`14171.4
`
`75757.6
`
`Fold
`enhancement
`
`1
`
`4
`4.4
`14.5
`80
`
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`FIG. 1. SDS-PAGE of protein fractions during the initial steps
`of the purification of the functional lysosomal sialic acid trans-
`porter from rat liver. Protein fractions were analyzed by SDS-PAGE
`(10% gel) and Coomassie Brilliant Blue R-250 stained. Per lane, an
`aliquot of approximately 30 mg of total protein was loaded. Lane 1,
`mid-range protein molecular weight markers (Promega). Lane 2, rat
`liver lysosomal membrane vesicles. Lane 3, Triton X-100-solubilized
`lysosomal membrane vesicles. Lane 4, 25 mM sodium phosphate eluate
`of first hydroxyapatite column. Lane 5, lentil lectin unretained fraction.
`Lane 6, 100 mM NaCl eluate of DEAE-Sephacel column.
`
`ture endoglycosidase F/N-glycosidase F. After treatment, the
`apparent molecular mass of the 57-kDa protein was not de-
`creased. 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 pro-
`tein 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-mercaptoeth-
`anol did not show any alteration of the electrophoretic behavior
`of the purified transport protein (data not shown). This indi-
`cates that the transporter is not functional as a (homo)dimer or
`polymer linked by disulfide bridges.
`Substrate Specificity of the Lysosomal Sialic Acid Transport-
`er—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 ly-
`sosomal 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.
`Interaction of the Lysosomal Sialic Acid Transporter with
`Iduronic Acid—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) (3, 4). The uronic acid iduronate (IdoA) represents,
`
`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 activ-
`ity over the starting material. Analysis of the protein composi-
`tion 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.
`2A). 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 (23). Another major 67-kDa protein represented the
`lysosomal membrane-bound subunit of acid phosphatase (24).
`The other proteins were considered as candidates for the lyso-
`somal sialic acid transporter.
`The next purification step consisted of a strong anion ex-
`change Mono Q column attached to the SMART system of
`Amersham Pharmacia Biotech. Retained proteins were eluted
`with a gradient of 0–210 mM NaCl. SDS-PAGE analysis by
`silver nitrate staining of the eluted proteins showed a predom-
`inant protein band with a molecular mass of ’57 kDa in the
`fractions 20/21 in which also the highest GlcA transport activ-
`ity was observed (Fig. 2B). 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. 2B).
`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 b-glucosi-
`dase 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.
`Properties of the Purified Lysosomal Sialic Acid Transport-
`er—To investigate the glycosylation of the transporter, the
`final protein preparation was incubated with the enzyme mix-
`
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`Purification of the Lysosomal Sialic Acid Transporter
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`TABLE II
`cis-Inhibition and trans-stimulation of [3H]GlcA uptake by mono-,
`di-, or tricarboxylic acids
`The partially purified (DEAE-Sephacel eluate) sialic acid transporter
`was reconstituted, and proteoliposomes were incubated 1 min at 37 °C
`with 10 mM [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 6 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 mM valinomycin and monensin. The trans-
`port assay was started by a 4-fold dilution in pH 5.5 incubation buffer
`with 2 mCi of [3H]GlcA and allowed to proceed for 1 min. In the samples
`that were preincubated without unlabeled compound, 0.25 mM unla-
`beled compound was added together with radiolabeled substrate to give
`the same extravesicular substrate concentration in both experiments.
`
`Transport activity
`
`trans-Stimulation
`
`pmol/mg/min % of control
`
`% of not trans-
`stimulated
`
`Control
`Acidic monosaccharides
`GlcA
`Neu5Ac
`IdoA
`Monocarboxylates
`Oxamate
`Pyruvate
`L-Lactate
`4-OH-butyrate
`Mevalonate
`Valproate
`Dicarboxylates
`Succinate
`Malate
`Malonate
`Maleate
`Fumarate
`a-Ketoglutarate
`Glutamate
`Tricarboxylate
`Citrate
`
`2280.5 6 132.6
`
`0
`0
`916.1 6 272.4
`
`553.0 6 45.5
`534.6 6 46.0
`195.6 6 67.8
`369.4 6 107.2
`510.0 6 24.0
`0
`
`0
`366.0 6 21.8
`685.3 6 37.5
`592.5 6 79.5
`234.3 6 81.0
`1090.2 6 199.6
`2437.8 6 195.4
`
`1665.9 6 30.6
`
`0
`0
`40
`
`24
`24
`8
`16
`22
`0
`
`0
`16
`30
`26
`10
`48
`107
`
`73
`
`200
`
`180
`
`150
`
`109
`
`79
`
`FIG. 3. Proton gradient-dependent uptake of [14C]L-lactate.
`Proteoliposomes of DEAE-Sephacel eluate prepared in 20 mM NaHepes,
`100 mM KCl, pH 7.4, were incubated with 15 mM [14C]L-lactate at 20 °C
`in 40 mM Mes (free acid), 10 mM valinomycin, pH 5.5 (with proton
`gradient, pHin 5 7.4 . pHout 5 5.5, l) or in 20 mM NaHepes, 10 mM
`valinomycin, pH 7.4 (no proton gradient, pHin 5 pHout 5 7.4, E).
`
`diolabeled [14C]L-lactate. The presence of an inwardly directed
`proton gradient (pHin 5 7.4 . pHout 5 5.5) stimulated initial
`uptake rates of lactate above equilibrium level (Fig. 3). At the
`top of overshoot, approximately 2% of external lactate was
`taken up inside the vesicles. This overshoot phenomenon was
`abolished in the absence of a proton gradient (pHin 5 pHout 5
`7.4), indicating that the transport of lactate is proton gradient-
`driven, similarly to the transport of the acidic monosaccharides
`Neu5Ac and GlcA. In these experiments, the use of low concen-
`
`FIG. 2. A 57-kDa protein correlates with the transport activity.
`Panel A shows the SDS-PAGE protein pattern of the preparation, which
`was applied to the Mono Q column. Panel B, bottom, elution profile of
`Mono Q column. Transport activity was measured in reconstituted
`proteoliposomes of the respective Mono Q fractions. Proteoliposomes
`(25 ml) were incubated 1 min at 37 °C with 10 mM [3H]GlcA in the
`presence of an inwardly directed proton gradient. Transport activity is
`expressed as pmol [3H]GlcA/min/25 ml. Top, SDS-PAGE and silver
`staining of corresponding Mono Q fractions.
`
`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 and trans-stimulation studies
`whether this uronic acid is also a substrate for the lysosomal
`sialic acid transporter (Table II). IdoA inhibited [3H]GlcA up-
`take, although less efficiently than Neu5Ac and GlcA. Further-
`more, 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.
`Interaction of the Lysosomal Sialic Acid Transporter with
`Small Monocarboxylates—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 their cis-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 the dicarboxylate transporters of the outer mito-
`chondrial membrane. The monocarboxylic and dicarboxylic ac-
`ids were all strong inhibitors, except for the amino acid gluta-
`mate and the Krebs cycle intermediate a-ketoglutarate.
`L-Lactate and the anti-epileptic drug valproic acid (dipropyl
`acetate), among the monocarboxylates, and succinate, among
`the dicarboxylates, were the strongest inhibitors (Table II). The
`tricarboxylate citrate showed no significant inhibition. To test
`whether inhibition represents interaction at the substrate
`binding site and consequently transport, we investigated the
`trans-stimulation effect of some representative mono- and di-
`carboxylate inhibitors on the uptake of [3H]GlcA. Partially
`purified protein preparations were reconstituted in proteolipo-
`somes and preloaded with an unlabeled compound at concen-
`trations of 1 mM, just above the Km of GlcA (0.4 mM) (6), and the
`uptake of [3H]GlcA was followed for 1 min. As shown in Table
`II, L-lactate as well as GlcA itself trans-stimulated the uptake
`of [3H]GlcA. Mevalonate and succinate did not cause trans-
`stimulation. Next, we investigated transport kinetics of L-lac-
`tate by the partially purified sialic acid transporter using ra-
`
`

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`34572
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`Purification of the Lysosomal Sialic Acid Transporter
`
`FIG. 4. Competitive inhibition of
`[3H]GlcA transport by L-lactate and
`of [14C]L-lactate transport by Neu5Ac.
`Initial proton-dependent transport rates
`of [3H]GlcA (1 min, 37 °C) and [14C]L-lac-
`tate (30 s, 20 °C) were measured in pro-
`teoliposomes of DEAE-Sephacel eluates.
`The uptake medium contained increasing
`concentrations of
`the respective sub-
`strates in the presence or absence of the
`inhibitors L-lactate or Neu5Ac. Data were
`plotted double reciprocally. A, [3H]GlcA
`uptake with (E) or without (l) cold 2 mM
`L-lactate. B, [14C]L-lactate uptake with
`(E) or without (l) cold 7 mM Neu5Ac.
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`trations of lactate (15 mM), lower temperature (20 °C), and the
`use of a pH far above the pK for the tested compounds limit the
`contribution of aspecific diffusion on the net uptake. Proton-
`driven [14C]L-lactate transport under apparent zero-trans con-
`ditions was saturable with a Km of approximately 0.4 mM and
`a Vmax of 500 nmol/30 s/mg protein. An Eadie-Hofstee plot of
`the kinetic data indicated a linear process, suggesting that only
`one type of transport system operates (data not shown).
`Although these studies provide evidence that the partially
`purified protein preparation is able to transport, in addition to
`acidic monosaccharides, many other small monocarboxylic ac-
`ids, it cannot be excluded that other proteins in this prepara-
`tion are present. However, the exchange of GlcA with L-lactate
`in the trans-stimulation experiments is strong evidence for
`transport of both compounds by the same protein. To provide
`further evidence that lactate and GlcA can be transported by
`the same carrier in the lysosomal membrane, competitive in-
`hibition experiments were performed. Proton-dependent trans-
`port of [3H]GlcA or [14C]L-lactate was measured in voltage
`clamped membranes with K1/valinomycin in the absence or
`presence of cold L-lactate or cold Neu5Ac as inhibitors, respec-
`tively. The results were fitted to a double reciprocal plot, show-
`ing a clear mode of competitive inhibition of lactate (calculated
`Ki of 2.5 mM) on GlcA transport and of Neu5Ac (calculated Ki of
`2 mM) on L-lactate transport (Fig. 4). Definite evidence that
`transport of lactate is performed by the lysosomal sialic acid
`transporter was obtained from cis-inhibition and concentration-
`dependent inhibition studies with the highly purified sialic acid
`transporter preparation. Proton-driven [3H]GlcA transport un-
`der apparent zero-trans conditions was completely inhibited in
`the presence of 7 mM unlabeled GlcA or Neu5Ac or L-lactate
`(Table III). Proton-driven [14C]L-lactate transport under appar-
`ent zero-trans conditions was inhibited totally by L-lactate and
`significantly by Neu5Ac. It is interesting to note that under
`these conditions GlcA did not inhibit. Because the GlcA trans-
`port assays are performed at 37 °C and the lactate transport
`assays at 20 °C, these apparent inconsistencies can be ex-
`plained by differences in affinities at different temperatures.
`Inhibition of [3H]GlcA transport by L-lactate was a clear con-
`centration-dependent process (Table III). Clearly, the highly
`purified transporter preparation contains a transporter that
`carries all three substrates, GlcA, Neu5Ac, and L-lactate (see
`also the above competitive inhibition experiments).
`Sensitivity to Covalent Protein Modifiers—In previous exper-
`iments studying the effect of protein modifiers on GlcA trans-
`port in native lysosomal membrane vesicles and reconstituted
`proteoliposomes, we have demonstrated the involvement of
`arginines (and possibly histidines) in substrate recognition (3,
`
`TABLE III
`cis-Inhibition of [3H]GlcA and [14C]L-lactate uptake by GlcA, Neu5Ac,
`and L-lactate in proteoliposomes of the highly purified lysosomal sialic
`acid transporter
`In the upper part of the table, the Mono Q eluate was reconstituted,
`and proteoliposomes were either incubated 1 min at 37 °C in the case of
`[3H]GlcA assay or 30 s at 20 °C in the case of [rosup;14C]L-lactate assay,
`both in the presence of an inwardly directed proton gradient and with
`7 mM of the indicated compounds. In the lower part of the table,
`proteoliposomes of the highly purified sialic acid transporter were in-
`cubated with 10 mM [3H]GlcA for 1 min at 37 °C in the presence of an
`inward directed proton gradient (pHin 5 7.4, pHout 5 5.5) and with
`v