Purification of the Lysosomal Sialic Acid Transporter

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 57kDa 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 that L-lactate is transported by the lysosomal sialic acid transporter. L-Lactate transport was proton gradient-dependent, saturable 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 transporter 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 transport 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 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 (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 chromosomal 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 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 (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)(8)(9).

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 Ϫ70°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
Reconstitution of the protein eluates into liposomes was performed as described earlier (6), with the following modification: proteoliposomes were formed by incubating the protein sample, containing detergent and phospholipid (total volume, 170 l), with 150 l 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 gradient. 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 l) were incubated at 37°C with 5 l of 240 mM Mes (free acid) containing 2 Ci of D-[1-3 H]GlcA (Amersham Pharmacia Biotech; specific activity, 6.6 Ci/mmol), resulting in an extravesicular pH of 5.5 and a final concentration of 10 M GlcA. Blank * 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. H]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 l 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 ϫ 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 Ci of [ 3 H]GlcA (final concentration, 10 M) in 40 mM Mes (free acid), resulting in an inwardly directed proton gradient (pH in ϭ 7.4 Ͼ pH out ϭ 5.5), and 7 mM of the tested compound.
For trans-stimulation studies, a 60% proteoliposome solution (25 l) 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 M monensin, 10 M valinomycin (Boehringer Mannheim) in the presence or absence of 1 mM unlabeled substrate. The assay was started by adding 75 l of an equivalent buffer containing 2 Ci of [ 3 H]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 (6).
The experiments with [ 14 C]L-lactate (Amersham Pharmacia Biotech; specific activity, 152 mCi/mmol) were largely performed as described for [ 3 H]GlcA. However, incubation mixtures contained 0.066 Ci of [ 14 C]Llactate (final concentration, 15 M) 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 sodium L-lactate and subtracted from all determinations. For protein side chain modification, proteoliposomes (100 l) 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 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 ϫ 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 material, Biogel HTP, Bio-Rad, packed by 15 s of tapping) at 4°C, with a maximum of 500 l 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 Na 2 HPO 4 , NaH 2 PO 4 , 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 l and desalted. A 50-l aliquot was used for the reconstitution assay and a 20-l 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 (10).
Step 3: Lentil Lectin-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 l and desalted (10). A 50-l aliquot was used for the reconstitution assay, and a 20-l aliquot was used for the protein assay.
Step 4: DEAE-Sephacel-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 Ϫ70°C.
Step 5: Hydroxyapatite-After the DEAE-eluate was thawed, a 0.5 ml sample was concentrated in a Centricon 10 device until 100 l and desalted (10). A 50 l aliquot was used for the reconstitution assay and a 20 l 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 Na 2 HPO 4 , NaH 2 PO 4 , 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 l and desalted (10). A 50-l aliquot was used for the reconstitution assay, and a 100-l aliquot was used for the protein assay.
Step 6: Mono Q-The concentrated hydroxyapatite eluate (150 l) 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 Na-Hepes, 100 mM KCl, 0.1% Triton X-100 by the desalting procedure as described above, and a 50-l 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 SDSpolyacrylamide gel electrophoresis according to Laemmli (11) of methanol/chloroform precipitated samples (12), 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 (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.

Purification of the Lysosomal Sialic Acid Transporter-Var-
ious membrane (transport) proteins have been successfully purified using hydroxyapatite as well as ion exchange chromatography in the presence of detergents (14 -18). In addition, affinity chromatography with oligosaccharide-specific lectins has been used to identify the major heavily glycosylated lysosomal membrane proteins: LAMPs (lysosomal-associated membrane proteins) and LIMPs (lysosomal integral membrane proteins) (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 proteoliposomes, and their transport activities were measured using 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 transporter was recovered from the column flow-through. Lentil lectin recognizes ␣-D-glucose and ␣-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 Ϸ14.5-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.  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 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 mM NaCl. SDS-PAGE analysis by silver nitrate staining of the eluted proteins showed a predominant protein band with a molecular mass of Ϸ57 kDa in the fractions 20/21 in which also the highest GlcA transport activity 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 ␤-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.
Properties of the Purified Lysosomal Sialic Acid Transporter-To investigate the glycosylation of the transporter, the final protein preparation was incubated with the enzyme mix-ture 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.
Substrate Specificity of the Lysosomal Sialic Acid Transporter-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.
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,  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 [ 3 H]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.
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 [ 3 H]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 mitochondrial membrane. The monocarboxylic and dicarboxylic acids were all strong inhibitors, except for the amino acid glutamate and the Krebs cycle intermediate ␣-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 dicarboxylate inhibitors on the uptake of [ 3 H]GlcA. Partially purified protein preparations were reconstituted in proteoliposomes and preloaded with an unlabeled compound at concentrations of 1 mM, just above the K m of GlcA (0.4 mM) (6), and the uptake of [ 3 H]GlcA was followed for 1 min. As shown in Table  II, L-lactate as well as GlcA itself trans-stimulated the uptake of [ 3 H]GlcA. Mevalonate and succinate did not cause transstimulation. Next, we investigated transport kinetics of L-lactate by the partially purified sialic acid transporter using ra-diolabeled [ 14 C]L-lactate. The presence of an inwardly directed proton gradient (pH in ϭ 7.4 Ͼ pH out ϭ 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 (pH in ϭ pH out ϭ 7.4), indicating that the transport of lactate is proton gradientdriven, similarly to the transport of the acidic monosaccharides Neu5Ac and GlcA. In these experiments, the use of low concen-   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 M [ 3 H]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 Ϯ 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 M valinomycin and monensin. The transport assay was started by a 4-fold dilution in pH 5.5 incubation buffer with 2 Ci of [ 3 H]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. trations of lactate (15 M), 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. Protondriven [ 14 C]L-lactate transport under apparent zero-trans conditions was saturable with a K m of approximately 0.4 mM and a V max 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 acids, it cannot be excluded that other proteins in this preparation 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 inhibition experiments were performed. Proton-dependent transport of [ 3 H]GlcA or [ 14 C]L-lactate was measured in voltage clamped membranes with K ϩ /valinomycin in the absence or presence of cold L-lactate or cold Neu5Ac as inhibitors, respectively. The results were fitted to a double reciprocal plot, showing a clear mode of competitive inhibition of lactate (calculated K i of 2.5 mM) on GlcA transport and of Neu5Ac (calculated K i 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 concentrationdependent inhibition studies with the highly purified sialic acid transporter preparation. Proton-driven [ 3 H]GlcA transport under apparent zero-trans conditions was completely inhibited in the presence of 7 mM unlabeled GlcA or Neu5Ac or L-lactate (Table III). Proton-driven [ 14 C]L-lactate transport under apparent 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 transport assays are performed at 37°C and the lactate transport assays at 20°C, these apparent inconsistencies can be explained by differences in affinities at different temperatures. Inhibition of [ 3 H]GlcA transport by L-lactate was a clear concentration-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 experiments studying the effect of protein modifiers on GlcA transport in native lysosomal membrane vesicles and reconstituted proteoliposomes, we have demonstrated the involvement of arginines (and possibly histidines) in substrate recognition (3,6). To investigate whether L-lactate transport is similarly affected by some of these protein modifiers, inhibition and substrate protection experiments were performed. 1 mM N-ethylmaleimide, a thiol-modifier, irreversibly inactivated transport of both GlcA (75% inhibition) and L-lactate (86% inhibition) in proteoliposomes from partially purified preparations. Phenylglyoxal, under these conditions an arginine modifier, significantly inhibited GlcA transport (44% of uninhibited rate). The inactivation of GlcA transport could be partially rescued when phenylglyoxal treatment was performed in the presence of the substrates GlcA, Neu5Ac, and L-lactate (96, 96 and 56% of uninhibited rate, respectively). Similarly, L-lactate transport was significantly inhibited by phenylglyoxal (59% of uninhibited rate) and protected in the presence of L-lactate, GlcA, and Neu5Ac (91, 88, and 71%, respectively, of uninhibited rate). Apparently, GlcA, Neu5Ac, and L-lactate all use the same substrate-binding site of the transport protein. DISCUSSION In this paper we describe the purification of the lysosomal sialic acid transport protein to apparent homogeneity based on its biological activity. To our knowledge, this is the first report  about the purification and detailed kinetic characterization of a lysosomal membrane carrier protein. Initial attempts using specific inhibitors like ␣-cyanocinnamate as ligands for affinity chromatography have not been successful. 2 Classical chromatographic techniques for membrane proteins were used. Hydroxyapatite chromatography was particularly successful in obtaining fractions enriched in transport activity. This medium has also been successful in the purification of several mitochondrial membrane transport proteins (14). Like some of those carriers, our transporter adsorbed weakly to hydroxyapatite (elution with 25 mM and 1 mM sodium phosphate buffer). This is probably due to a large micellar shell around the protein formed by Triton X-100 molecules. Silver-stained SDS-PAGE gels showed only one protein band with a molecular mass of approximately 57 kDa, correlating with the transport activity of GlcA (Fig. 2B). Concerning the enrichment of this lysosomal membrane transporter and the highly sensitive silver staining, it is unlikely that the activity resides in another minor, not visualized protein. Apparently, the more abundant and well characterized integral lysosomal membrane proteins, such as LAMPs and LIMPs, are densely N-glycosylated glycoproteins (22). Based on the deglycosylation studies, the sialic acid transporter appears not to be heavily glycosylated. For several known anion carriers N-glycosylation is not required for their transport function (25). The same is true for some other membrane transporters (26,27). Some sugar carriers are known to be functional because homodimers possibly linked by S-S bridges (28,29). In contrast, our transporter does not have a quaternary structure in which subunits are held together by S-S bridges.
In previous studies, we showed that the structural requirements for recognition of the substrate by the carrier were different from those of other proteins involved in sialic acid metabolism (lysosomal sialidase, the Golgi system transporter for CMP-sialic acid and sialyltransferases). For the carrier, the C-1 carboxylic group had to be unsubstituted as well as the native hydroxyl group at C-2 (3). In addition, the kinetic properties of the lysosomal sialic acid transporter were similar to those of other known proton cotransporters for other organic anions (3,6). Structurally different monosaccharides, such as sialic acid (N-acetylneuraminic acid) (a 9-carbon N-substituted monosaccharide), glucuronic acid (a hemiacetal-forming acid monosaccharide with a C-6 carboxylic group), and gluconic acid (a non-hemiacetal-forming acid monosaccharide with a C-1 carboxylic group) are all recognized by this carrier (3,6). In this paper we extended the group of acidic monosaccharide substrates with IdoA, like GlcA an important physiological component of mammalian glycosaminoglycans. The lysosomal sialic acid transporter is the only mammalian transporter known to allow transport of uronic acids, aldonic acids, and N-substituted neuraminic acids (3,4). The physiological role of sialic acid and glucuronic acid transport has earlier been demonstrated by the accumulation of these monosaccharides and by their defective transport in lysosomes of patients with sialic acid storage diseases (4,30). So far, lysosomal accumulation of IdoA in these patients has not been investigated. The contribution of the different acidic monosaccharides to the pathology remains therefore elusive.
Because aliphatic monocarboxylates like pyruvate inhibit transport (3), we compared the biochemical characteristics of the purified carrier with those of the aliphatic monocarboxylate transporters. To date, several monocarboxylate transporters present in the plasma membrane of various mammalian cells have been cloned and characterized: MCT1, MCT2, and MCT3 (7)(8)(9)31). They are proton-coupled transporters with a broad specificity for short chain monocarboxylates (including lactate and pyruvate), showing differences in cellular distribution. A number of non-sugar mono-and dicarboxylic acids were effective inhibitors of initial uptake rates of GlcA transport by the partially purified lysosomal sialic acid transporter (Table II). The inhibition observed was not dependent on changes in membrane potential, because experiments were performed in the presence of K ϩ and valinomycin. These monocarboxylates are transported by the MCTs with different affinities, except mevalonate (7). Mevalonate is transported by Mev, a homologue of MCT1 from Chinese hamster ovary cells (32), but is not transported efficiently by MCT1 (8). trans-Stimulation experiments demonstrated that mevalonate is not transported by the lysosomal sialic acid transporter (Table II). Most of the tested dicarboxylates are known to be substrates of the dicarboxylate carrier in mitochondria (e.g. succinate, malate, and malonate) (33). For the di-and tricarboxylic acids glutamate, ␣-ketoglutarate, and citrate specific mitochondrial transport proteins have been identified (34). The lack of trans-stimulation in our experiments suggested that one of the strongest dicarboxylate inhibitors (i.e. succinate) is not transported by the lysosomal sialic acid transporter. Similarly, a lactate transporter in placental brush border membrane vesicles is unable to transport the cis-inhibitor succinate (35). Transport of [ 3 H]GlcA by the partially purified lysosomal sialic acid transporter is cis-inhibited and trans-stimulated by the small aliphatic monocarboxylate L-lactate. This inhibition of [ 3 H]GlcA transport by L-lactate showed a competitive mode. The same cis-inhibition of [ 3 H]GlcA transport by L-lactate was observed using the highly purified preparation, containing one major protein band on SDS-PAGE. This inhibition was clearly dependent on the concentration of the inhibitor L-lactate. Transport of [ 14 C]L-lactate by the partially purified lysosomal sialic acid transporter preparation is a proton gradient-mediated process that can be competitively inhibited by Neu5Ac but surprisingly not by GlcA. [ 14 C]L-lactate transport by the highly purified preparation also showed clear inhibition by Neu5Ac but not by GlcA. Apparently, at 37°C ([ 3 H]GlcA transport assay) as well as GlcA, Neu5Ac, and L-lactate are recognized by the transporter, but at 20°C ([ 14 C]L-lactate transport assay) the affinity for L-lactate and Neu5Ac is much higher than for GlcA. This can be explained by structural differences among these molecules, especially at the site of the carboxyl group. Another possible explanation would be the presence of different lactate transporters, one of which is not fully inhibited by the other two compounds. However, kinetic analysis (linear Eadie-Hofstee plots) did not show any evidence for different lactate transporters. In conclusion, all these data provide strong evidence that the lysosomal sialic acid transporter is able to transport besides acidic monosaccharides also other short chain monocarboxylates like L-lactate. The competitive inhibition experiments, the trans-stimulation experiments, and the cis-inhibition experiments using the highly purified preparation exclude the possibility that the observed L-lactate transport is due to a contaminating protein.
We do not know whether L-lactate is a physiological substrate in the lysosome. However, a probenecid-inhibitable organic anion transporter, possibly also recognizing lactate, was postulated in endosomes from macrophages (36). Probenecid is also an inhibitor of the sialic acid transporter in native lysosomal membranes. 3 Proton cotransport of L-lactate has been described for MCT1, MCT2, and MCT3 by kinetic analysis and by inhibition with para-chloromercuribenzoic acid (8,9,31). Lac-tate and pyruvate show a structural similarity with Neu5Ac, of which pyruvate is the natural precursor. Although it would be interesting to investigate whether MCT1 and/or MCT2 also could transport monocarboxylic monosaccharides, it is assumed that MCT1 transports mainly unbranched aliphatic monocarboxylates from C-2 to C-5 but not mevalonate. [ 3 H]GlcA uptake is cis-inhibited by the anti-epileptic drug valproic acid. Valproic acid is also a known inhibitor of the mitochondrial pyruvate carrier (37) and of MCT1 in Chinese hamster ovary cells (38), suggesting a similarity between the lysosomal system here investigated and these monocarboxylate carriers. It would be interesting to study in more detail the role of the lysosomal sialic acid transporter in the transport of valproic acid, its metabolites, and other organic anions by future experiments using their radioactive forms.
Both the lysosomal sialic acid transporter and MCTs are sensitive to hydroxycinnamic acid derivatives, the stilbene disulfonate DIDS, and para-chloromercuribenzoic acid (6 -9). However, the difference in sensitivity to the amino acid modifier N-ethylmaleimide distinguishes MCT1 from the lysosomal sialic acid transporter. MCT1 is only poorly inhibited by Nethylmaleimide (7) in contrast to the sialic acid transporter, which is strongly inhibited by N-ethylmaleimide (this paper). Another difference between the sialic acid transporter and MCT1 is the lower K m for lactate transport, 0.4 mM (this paper) versus 8 mM for MCT1 (9). Recently, Price et al. have cloned and sequenced four new human MCT homologues, MCT4 to MCT7 (39). Their similarities and some strongly conserved sequence motifs provide evidence for an ancient family of transporters. Considering that the conversion of Phe 360 into Cys changes MCT1 from a lactate transporter to a mevalonate transporter (8,32), it is possible that minimal structural differences among MCTs account for major differences in substrate specificity. The amino acid modifier phenylglyoxal inactivated the reconstituted sialic acid transporter. This reagent revealed essential arginine/histidine residues at the substrate-binding site of the transporter, because its inactivation could be prevented by concomitant incubation with Neu5Ac or GlcA (6). L-Lactate acts on the same substrate-binding site as GlcA, because the inhibitory effect of phenylglyoxal on the uptake of [ 14 C]L-lactate could be prevented by concomitant incubation with L-lactate or GlcA (this paper). Apparently, phenylglyoxal modifies essential arginine residues present in the lactate-binding site of the carrier protein. Also, for the MCTs an essential function for a conserved Arg residue has been proposed (39). In conclusion, we have observed an overlap in substrate specificity for monocarboxylates, a similar proton cotransport mechanism (with consequently the sensitivity to para-chloromercuribenzoic acid), a similar sensitivity to hydroxycinnamic acid derivatives and stilbene disulfonates, and the presence of essential arginine residues in the substrate binding sites of the sialic acid carrier and the monocarboxylate carriers. Altogether, these data suggest functional and structural similarities of our transporter with previously characterized monocarboxylate transporters.
The lysosomal transport mechanism for sialic acid is defective in different clinical variants of the neurodegenerative disorder sialic acid storage disease. We demonstrated that the purified sialic acid transporter interacts with lactate, an important energy source in brain. Further studies of lactate metabolism in brain from these patients might help to understand events of neurodegeneration in this disorder. By NMR spectroscopy, it is possible to show abnormalities in metabolites in brain (40). We are not aware of NMR spectroscopy studies in sialic acid storage diseases, which could be applied to study lactate metabolism in brain of these patients.
By N-terminal sequencing, we have obtained a stretch of amino acids from the 57-kDa protein, but still too many doubtful residues are present, making gene cloning difficult. However, the functional and possible structural homology with monocarboxylate transporters may provide a tool to identify and clone the sialic acid transporter gene by screening for homologues in available sequence libraries.