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.

Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.

Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).

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.

Isolation and structural characterization of sialic acid-containing storage material from mucolipidosis I (sialidosis) fibroblasts.

Sialic acid-containing storage material was isolated from cultured human mucolipidosis I (sialidosis) fibroblasts by gel permeation chromatography on Bio-Gel P-6 followed by medium-pressure anion-exchange chromatography on Mono Q. The structure determination of the isolated sialyloligosaccharides was carried out by 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis and analytical HPLC. The storage material showed completely sialylated mono-, di- and triantennary N-glycosidic N-acetyllactosamine oligosaccharides having the Man beta 1----4GlcNAc sequence at the reducing end in common. Heterogeneity occurred with respect to the linkages between terminal sialic acid and the penultimate galactose residues (alpha 2----3/alpha 2----6). It turned out that all the identified carbohydrate chains are consistent with the neuraminidase deficiency.

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis.

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.

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.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases.

OBJECTIVE: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS). METHODS: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. RESULTS: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L). CONCLUSION: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Brain abnormalities in a case of malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) deficiency is an extremely rare inborn error of metabolism that presents with metabolic acidosis, hypoglycemia, and/or cardiomyopathy. Patients also show neurological signs and symptoms that have been infrequently reported. We describe a girl with MCD deficiency, whose brain MRI shows white matter abnormalities and additionally diffuse pachygyria and periventricular heterotopia, consistent with a malformation of cortical development. MLYCD-gene sequence analysis shows normal genomic sequence but no messenger product, suggesting an abnormality of transcription regulation. Our patient has strikingly low appetite, which is interesting in the light of the proposed role of malonyl-CoA in the regulation of feeding control, but this remains to be confirmed in other patients. Considering the incomplete understanding of the role of metabolic pathways in brain development, patients with MCD deficiency should be evaluated with brain MRI and unexplained malformations of cortical development should be reason for metabolic screening.

Homozygosity for the p.K136E mutation in the SLC17A5 gene as cause of an Italian severe Salla disease.

Lysosomal free sialic acid storage diseases are recessively inherited allelic neurodegenerative disorders that include Salla disease (SD) and infantile sialic acid storage disease (ISSD) caused by mutations in the SLC17A5 gene encoding for a lysosomal membrane protein, sialin, transporting sialic acid from lysosomes. The classical form of SD, enriched in the Finnish population, is related to the p.R39C designed Salla(FIN) founder mutation. A more severe phenotype is due both to compound heterozygosity for the p.R39C mutation and to different mutations. The p.R39C has not been reported in ISSD. We identified the first case of SD caused by the homozygosity for p.K136E (c.406A>G) mutation, showing a severe clinical picture, as demonstrated by the early age at onset, the degree of motor retardation, the occurrence of peripheral nerve involvement, as well as cerebral hypomyelination. Recently, in vitro functional studies have shown that the p.K136E mutant produces a mislocalization and a reduced activity of the intracellular sialin. We discuss the in vivo phenotypic consequence of the p.K136E in relation to the results obtained by the in vitro functional characterization of the p.K136E mutant. The severity of the clinical picture, in comparison with the classical SD, may be explained by the fact that the p.K136E mutation mislocalizes the protein to a greater degree than p.R39C. On the other hand, the presence of a residual transport activity may account for the absence of hepatosplenomegaly, dysostosis multiplex, and early lethality typical of ISSD and related to the abolished transport activity found in this latter form.

Purification and partial characterization of lysosomal neuraminidase from human placenta.

Lysosomal neuraminidase and beta-galactosidase are present in a complex together with a 32-kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. beta-Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non-dissociated beta-galactosidase/protective protein complex using beta-galactosidase-specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS-PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32-kDa protective protein was dissociated from the beta-galactosidase/protective protein complex, and purified. Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with beta-galactosidase and protective protein. By immunoblotting evidence is provided that the 76-kDa protein is a subunit of neuraminidase which, in association with the 32-kDa protective protein, is essential for neuraminidase activity.

Free N-acetylneuraminic acid (NANA) storage disorders: evidence for defective NANA transport across the lysosomal membrane.

To study the biochemical defect underlying N-acetylneuraminic acid (NANA) storage disorders (NSD), a tritium-labeled NANA-methylester was prepared and its metabolism was studied in normal and mutant human fibroblasts. The uptake of methylester, its conversion into free NANA, and the release of free NANA was studied in lysosome-enriched fractions. In three clinically different types of NSD accumulation of free NANA was observed and the half-life of this compound was significantly increased. Our observations indicate the existence of a transport system for NANA across the lysosomal membrane, which is deficient in all variants of NSD.

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.

The relation between human lysosomal beta-galactosidase and its protective protein.

Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.

Lysosomal transport disorders.

In the group of lysosomal storage diseases, transport disorders occupy a special place because they represent rare examples of inborn errors of metabolism caused by a defect of an intracellular membrane transporter. In particular, two disorders are caused by a proven defect in carrier-mediated transport of metabolites: cystinosis and the group of sialic acid storage disorders (SASD). The recent identification of the gene mutations for both disorders will improve patient diagnosis and shed light on new physiological mechanisms of intracellular trafficking.

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.

Complementation analysis of human sialidase deficiency using natural substrates.

Complementation analysis by somatic cell hybridization to produce heterokaryons has shown that at least three complementation groups exist within the disorders in which the enzyme sialidase is deficient. We have confirmed these results by electrophoretic analysis of two glycoprotein enzymes, adenosine deaminase and acid phosphatase, which show aberrant electrophoretic mobilities in these disorders. These abnormal forms, which have excess sialic acid bound, disappear on complementation and are replaced by normal mobility components. It is suggested that the sialidase produced on complementation uses the abnormal forms as natural substrates and that they may represent normal intermediates in the processing of glycoprotein enzymes.

Human placental neuraminidase. Activation, stabilization and association with beta-galactosidase and its protective protein.

Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37 degrees C and acid pH. Using beta-galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with beta-galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of beta-galactosidase. The formation of multimeric forms of beta-galactosidase is known to require a 32000-Mr 'protective' protein. Monospecific antibodies against this 'protective' protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000-Mr beta-galactosidase protein and against the 32000-Mr 'protective' protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000-Mr 'protective' protein is present both in association with the beta-galactosidase multimer and with the high-density multimeric form together with neuraminidase. Our data further suggest that association of the 32000-Mr 'protective' protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000-Mr 'protective' protein is known to be absent.