A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases.

Sialic acid storage diseases (SASD, MIM 269920) are autosomal recessive neurodegenerative disorders that may present as a severe infantile form (ISSD) or a slowly progressive adult form, which is prevalent in Finland (Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation; visceromegaly and coarse features are also present in infantile cases. Progressive cerebellar atrophy and dysmyelination have been documented by magnetic resonance imaging (ref. 4). Enlarged lysosomes are seen on electron microscopic studies and patients excrete large amounts of free sialic acid in urine. A H+/anionic sugar symporter mechanism for sialic acid and glucuronic acid is impaired in lysosomal membranes from Salla and ISSD patients. The locus for Salla disease was assigned to a region of approximately 200 kb on chromosome 6q14-q15 in a linkage study using Finnish families. Salla disease and ISSD were further shown to be allelic disorders. A physical map with P1 and PAC clones was constructed to cover the 200-kb area flanked by the loci D6S280 and D6S1622, providing the basis for precise physical positioning of the gene. Here we describe a new gene, SLC17A5 (also known as AST), encoding a protein (sialin) with a predicted transport function that belongs to a family of anion/cation symporters (ACS). We found a homozygous SLC17A5 mutation (R39C) in five Finnish patients with Salla disease and six different SLC17A5 mutations in six ISSD patients of different ethnic origins. Our observations suggest that mutations in SLC17A5 are the primary cause of lysosomal sialic acid storage diseases.

Sialic acid storage diseases. A multiple lysosomal transport defect for acidic monosaccharides.

A defective efflux of free sialic acid from the lysosomal compartment has been found in the clinically heterogeneous group of sialic acid storage disorders. Using radiolabeled sialic acid (NeuAc) as a substrate, we have recently detected and characterized a proton-driven carrier for sialic acid in the lysosomal membrane from rat liver. This carrier also recognizes and transports other acidic monosaccharides, among which are uronic acids. If no alternative routes of glucuronic acid transport exist, the disposal of uronic acids can be affected in the sialic acid storage disorders. In this study we excluded the existence of more than one acidic monosaccharide carrier by measuring uptake kinetics of labeled glucuronic acid [( 3H]GlcAc) in rat lysosomal membrane vesicles. [3H]GlcAc uptake was carrier-mediated with an affinity constant of transport (Kt) of 0.3 mM and the transport could be cis-inhibited or trans-stimulated to the same extent by sialic acid or glucuronic acid. Human lysosomal membrane vesicles isolated from cultured fibroblasts showed the existence of a similar proton-driven transporter with the same properties as the rat liver system (Kt of [3H]GlcAc uptake 0.28 mM). Uptake studies with [3H]NeuAc and [3H]GlcAc in resealed lysosome membrane vesicles from cultured fibroblasts of patients with different clinical presentation of sialic acid storage showed defective carrier-mediated transport for both sugars. Further evidence that the defective transport of acidic sugars represents the primary genetic defect in sialic acid storage diseases was provided by the observation of reduced, half-normal transport rates in lymphoblast-derived lysosomal membrane vesicles from five unrelated obligate heterozygotes. This study reports the first observation of a human lysosomal transport defect for multiple physiological compounds.

DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways.

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

Transport of organic anions by the lysosomal sialic acid transporter: a functional approach towards the gene for sialic acid storage disease.

Transport of sialic acid through the lysosomal membrane is defective in the human sialic acid storage disease. The mammalian sialic acid carrier has a wide substrate specificity for acidic monosaccharides. Recently, we showed that also non-sugar monocarboxylates like L-lactate are substrates for the carrier. Here we report that other organic anions, which are substrates for carriers belonging to several anion transporter families, are recognized by the sialic acid transporter. Hence, the mammalian system reveals once more novel aspects of solute transport, including sugars and a wide array of non-sugar compounds, apparently unique to this system. These data suggest that the search for the sialic acid storage disease gene can be initiated by a functional selection of genes from a limited number of anion transporter families. Among these, candidates will be identified by mapping to the known sialic acid storage disease locus.

Characterization of a heavy metal ion transporter in the lysosomal membrane.

Lysosomes are thought to play a role in various aspects of heavy metal metabolism. In the present study we demonstrate for the first time the presence of a heavy metal ion transport protein in the lysosomal membrane. Uptake of radioactive silver both in highly purified lysosomal membrane vesicles and in purified intact lysosomes showed the typical kinetics of a carrier-mediated process. This transport was stimulated by ATP hydrolysis, and showed specificity for Ag+, Cu2+, and Cd2+. All biochemical properties of this lysosomal metal ion transporter could classify it as a heavy metal transporting P-type ATPase. Long Evans Cinnamon (LEC) rats, an animal model for the copper transport disorder Wilson disease, showed normal lysosomal silver transport.

Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses.

Lysosomal membrane vesicles isolated from rat liver were exploited to analyze the mechanism of glucose transport across the lysosomal membrane. Uptake kinetics of [14C]D-glucose showed a concentration-dependent saturable process, typical of carrier-mediated facilitated transport, with a Kt of about 75 mM. Uptake was unaffected by Na+ and K+ ions, membrane potentials, and proton gradients but showed an acidic pH optimum. Lowering the pH from 7.4 to 5.5 had no effect on the affinity of the carrier for the substrate but increased the maximum rate of transport about 3-fold. As inferred from the linearity of Scatchard plots, a single transport mechanism could account for the uptake of glucose under all conditions tested. As indicated by the transstimulation properties of the carrier, other neutral monohexoses, including D-galactose, D-mannose, D- and L-fucose were transported by this carrier. The transport rates and affinities of these sugars, measured by the use of their radiolabeled counterparts, were in the same range as those for D-glucose. Pentoses, sialic acid, and other acidic monosaccharides including their lactones, aminosugars, N-acetyl-hexosamines, and most L-stereoisomers, particularly those not present in mammalian tissues, were not transported by this carrier. Glucose uptake and transstimulation were inhibited by cytochalasin B and phloretin. The biochemical properties of this transporter differentiate it from other well-characterized lysosomal sugar carriers, including those for sialic acid and N-acetylhexosamines. The acidic pH optimum of this glucose transporter is a unique feature not shared with any other known glucose carrier and is consistent with its lysosomal origin.

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized proteins were incorporated in egg yolk phospholipids. The reconstituted proteoliposomes show proton-driven carrier-mediated uptake of acidic monosaccharides. The reconstituted carrier was compared in several characteristics with the transporter as present in the native lysosomal membrane. Transporter substrate affinity (Kt for glucuronic acid = 0.4 mM) and specificity for acidic monosaccharides are completely retained. The proteoliposomes also demonstrate trans-stimulation properties with both substrates sialic acid and glucuronic acid. The transporter is inhibited, both in its native and in the reconstituted state, by the sulfhydryl-modifying agents p-chloromercuribenzoic acid, N-ethylmaleimide, and phenyl isothiocyanate. In native membrane vesicles, arginine and histidine modifiers phenylglyoxal and diethyl pyrocarbonate inactivated transport in a substrate-protectable manner. In reconstituted proteoliposomes, similar inhibition was observed. However, protection by substrates was achieved only after treatment with phenylglyoxal. These data suggest that arginine or histidine residues or both are present at or near the substrate binding site of the carrier. Possibly, other essential histidines become exposed in the reconstituted state. The successful functional reconstitution of the lysosomal sialic acid carrier represents an important step towards its purification and its detailed molecular characterization.

Sialic acid storage disorders: observations on clinical and biochemical variation.

Lysosomal accumulation of free sialic acid results in two phenotypically distinct inherited metabolic disorders, Salla disease and infantile sialic acid storage disease. Clinical and biochemical findings in both diseases are reviewed. Recent studies indicate that sialic acid storage is a consequence of defective function of a lysosomal membrane transport system specific for sialic acid and some other acidic monosaccharides.

Purification of the lysosomal sialic acid transporter. Functional characteristics of a monocarboxylate 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 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 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).

Fibroblast silver loading for the diagnosis of Menkes disease.

Menkes disease is a genetic disorder of copper metabolism. Copper uptake and retention assays on fibroblast or amniotic fluid cell cultures have been used for pre- and postnatal diagnosis. These copper loading tests are complicated by the use of 64Cu, which is not commonly available and has a very short (12.8 hours) physical half life. Besides copper, silver is also a substrate for the bacterial homologue of the Menkes transport protein. We report here that loading tests using radioactive silver (110mAg), instead of copper, can be used for the diagnosis of Menkes disease. 110mAg is commercially available and has a convenient physical half life of 250 days, which makes it suitable for use in diagnostic laboratories. Our studies support the hypothesis that reduction of divalent to monovalent copper is an essential step preceding transport.

Nucleotide-activated chloride channels in lysosomal membranes.

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.