Varied Mechanisms Underlie the Free Sialic Acid Storage Disorders

Abstract

Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462–465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832–840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases. Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462–465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832–840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases. Lysosomes are intracellular acidic organelles filled with hydrolytic enzymes. They are primarily responsible for the compartmentalized degradation of macromolecules as part of an intrinsic turnover process and as a mechanism for processing endocytosed nutrients. The importance of these organelles in normal cellular functions is apparent from the phenotype of a group of degenerative diseases designated as lysosomal storage disorders (3Schiffmann R. Brady R.O. Drugs. 2002; 62: 733-742Crossref PubMed Scopus (91) Google Scholar, 4Moser H.W. Curr. Opin. Neurol. Neurosurg. 1992; 5: 355-358PubMed Google Scholar). The hallmark of these disorders is the accumulation of material within enlarged lysosomes. The typical clinical phenotype is one of a progressive multiorgan involvement with a predominance of neurological dysfunction and often early death. For many of these diseases genetic studies have identified causative mutations, most of which occur in genes encoding enzymes involved in the hydrolysis of macromolecules. The major products of the enzymatic processes in lysosomes are small organic molecules. While the membrane-bound nature of the organelle allows for hydrolytic enzymes to be isolated from the cytosol, it also requires mechanisms for the release of these end products. Biochemical studies have identified several lysosomal transport systems that facilitate the movement of amino acids, nucleotides, lipids, vitamins, and sugars across the lysosomal membrane (5Pisoni R.L. Thoene J.G. Biochim. Biophys. Acta. 1991; 1071: 351-373Crossref PubMed Scopus (94) Google Scholar, 6Mancini G.M. Havelaar A.C. Verheijen F.W. J. Inherit. Metab Dis. 2000; 23: 278-292Crossref PubMed Scopus (45) Google Scholar). However, the molecular identification of the lysosomal transport systems has been difficult. Their intracellular localization and the presence of lysosomes or an equivalent organelle in all eukaryotic cells have hampered the use of expression cloning. Genetic studies of lysosomal storage disorders have offered some insight into the molecular mechanisms of the transport processes. For example, the gene mutated in cystinosis has been shown to encode a lysosomal cystine transporter (7Town M. Jean G. Cherqui S. Attard M. Forestier L. Whitmore S.A. Callen D.F. Gribouval O. Broyer M. van't Bates G.P. Hoff W. Antignac C. Nat. Genet. 1998; 18: 319-324Crossref PubMed Scopus (466) Google Scholar), while a fatty acid transporter (NPC1) localized to lysosomes and endosomes has been shown to be the product of a gene mutated in Niemann-Pick disease Type C (8Davies J.P. Chen F.W. Ioannou Y.A. Science. 2000; 290: 2295-2298Crossref PubMed Scopus (255) Google Scholar, 9Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Science. 1997; 277: 232-235Crossref PubMed Scopus (698) Google Scholar). Recent evidence also indicates that CLN3, the protein mutated in one form of Batten Disease, mediates a lysosomal arginine-H+ antiport activity (10Kim Y. Ramirez-Montealegre D. Pearce D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15458-15462Crossref PubMed Scopus (90) Google Scholar). Salla disease and infantile sialic acid storage disorder (ISSD) 1The abbreviations used are: ISSD, infantile sialic acid storage disorder; EST, expressed sequence tag; HA, hemagglutinin; AP, adaptor protein; MES, 4-morpholineethanesulfonic acid; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; ER, endoplasmic reticulum; PDI, protein disulfide-isomerase.1The abbreviations used are: ISSD, infantile sialic acid storage disorder; EST, expressed sequence tag; HA, hemagglutinin; AP, adaptor protein; MES, 4-morpholineethanesulfonic acid; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; ER, endoplasmic reticulum; PDI, protein disulfide-isomerase. are autosomal recessive lysosomal storage disorders with common features but differing degrees of severity (1Verheijen F.W. Verbeek E. Aula N. Beerens C.E. Havelaar A.C. Joosse M. Peltonen L. Aula P. van der Galjaard H. Spek P.J. Mancini G.M. Nat. Genet. 1999; 23: 462-465Crossref PubMed Scopus (218) Google Scholar, 2Aula N. Salomaki P. Timonen R. Verheijen F. Mancini G. Mansson J.E. Aula P. Peltonen L. Am. J. Hum. Genet. 2000; 67: 832-840Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Salla Disease is characterized by developmental delay with marked cognitive and motor impairment noticeable at 6–12 months of age. However, many affected individuals reach adulthood (11Varho T.T. Alajoki L.E. Posti K.M. Korhonen T.T. Renlund M.G. Nyman S.R. Sillanpaa M.L. Aula P.P. Pediatr. Neurol. 2002; 26: 267-273Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). ISSD has a more severe phenotype with intrauterine hydrops, neonatal ascites, dysmorphic features, and death by 2 years of age. The cellular pathology for both diseases consists of enlarged lysosomes filled with high concentrations of free sialic acid, an amino sugar related to neuraminic acid (12Aula P. Autio S. Raivio K.O. Rapola J. Thoden C.J. Koskela S.L. Yamashina I. Arch. Neurol. 1979; 36: 88-94Crossref PubMed Scopus (107) Google Scholar, 13Virtanen I. Ekblom P. Laurila P. Nordling S. Raivio K.O. Aula P. Pediatr. Res. 1980; 14: 1199-1203Crossref PubMed Scopus (19) Google Scholar, 14Renlund M. Aula P. Raivio K.O. Autio S. Sainio K. Rapola J. Koskela S.L. Neurology. 1983; 33: 57-66Crossref PubMed Google Scholar). The free sialic acid is derived from the oligosaccharide chains of sialylated glycoproteins and glycolipids that have been degraded in the lysosme. Previous studies have demonstrated that individuals with Salla disease and ISSD accumulate free sialic acid in their lysosomes due to a defect in a proton-coupled sialic acid transport activity (14Renlund M. Aula P. Raivio K.O. Autio S. Sainio K. Rapola J. Koskela S.L. Neurology. 1983; 33: 57-66Crossref PubMed Google Scholar, 15Renlund M. Tietze F. Gahl W.A. Science. 1986; 232: 759-762Crossref PubMed Scopus (101) Google Scholar, 16Tietze F. Seppala R. Renlund M. Hopwood J.J. Harper G.S. Thomas G.H. Gahl W.A. J. Biol. Chem. 1989; 264: 15316-15322Abstract Full Text PDF PubMed Google Scholar). Despite clear differences in the severity of the clinical phenotypes, both forms of the disease are associated with no detectable lysosomal sialic acid transport. Genetic studies of individuals with Salla Disease led to the determination that a gene encoding a protein designated sialin contains the causative mutations (1Verheijen F.W. Verbeek E. Aula N. Beerens C.E. Havelaar A.C. Joosse M. Peltonen L. Aula P. van der Galjaard H. Spek P.J. Mancini G.M. Nat. Genet. 1999; 23: 462-465Crossref PubMed Scopus (218) Google Scholar). Sialin is a member of the anion/cation symporter (ACS) family of proteins and has a high degree of similarity to the vesicular glutamate transporters (VGUT1–3) (17Reimer R.J. Edwards R.H. Pfluegers Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar). To date 16 mutations in sialin have been associated with the disease phenotype (2Aula N. Salomaki P. Timonen R. Verheijen F. Mancini G. Mansson J.E. Aula P. Peltonen L. Am. J. Hum. Genet. 2000; 67: 832-840Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The most common mutation (R39C) is found in Salla disease. The more severe ISSD is associated with mutations a and mutations that lead to early or To the mechanisms by which mutations lead to we to the function of a lysosomal we are to the protein on the for the use of to transport this we directly demonstrate that sialin is a sialic acid transporter with characteristics similar to of the native lysosomal transport system. the effect of mutations on transport activity we a direct correlation between the degree of transport activity and the severity of the clinical In addition, we demonstrate that one mutation to of the protein in the endoplasmic reticulum, that altered trafficking of sialin also to the of these of Sialin and predicted sialin sequence used to expressed sequence in the sequence to the and of the sequence and high used to the sequence from The into the expression To mutations and for with the the of the The to a sequence for to of the To facilitate the of a of an into the of the protein in a mutation which the localization or function of the protein. and cells in with at in and all and To in used to the of the For of used with the to that by the of cells at in the cells as and the with as A. 2002; Full Text Full Text PDF PubMed Scopus Google and on The used at and the used at with or at for used for cells on at a of and the as transport with acid as J. C. R.J. Edwards R.H. J. 2003; 23: PubMed Google Scholar). For a lysosomal transporter characterized with J. C. R.J. Edwards R.H. J. 2003; 23: PubMed Google Scholar), used and the for and the For the is the of For all are from with indicating the of the of in the of a with and of with to and from at the cells with of and in of 2 in for on of with to the the proteins isolated on as 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). on an to and as R.J. Edwards R.H. J. J. 1998; 18: PubMed Google Scholar). of with and of the to for to The used at and the used at of activity, transport activity and analysis of expression in for the and with the at as For of protein expression and to a for of identified and in The in a with a from cells the protein as and from all activity for from the by by expression expression with Disease in facilitate the of the of sialin we identified a of the protein. of led to the identification of to the and of the sialin to a sequence to the of the of the sequence a that a acid with and and similarity to the protein. similar degree of is also in of the or to the identified or of the proteins indicates that the disease-causing mutations occur the protein with no The predicted that the mutations occur in within and or In the amino in the are in the of a The associated with the mutations are the the is a single in the of one The of analysis of sialin or related proteins the of the mutations on their in the protein. of to of transport activity of recombinant sialin is by the that the protein is expressed in all cells of eukaryotic To the of lysosomal sialic acid transport activity we to a form of sialin that to the for to activity of the recombinant protein. While a membrane sialic acid transport activity has been S.R. C. PubMed Scopus Google Scholar), we found no in that this be In sialin from we a sequence in the amino that is from to with an acidic amino acid is a for adaptor protein 2003; PubMed Scopus Google Scholar). with or are for of some lysosomal membrane proteins 2003; PubMed Scopus Google Scholar), we to this in sialin is involved in We the of the with the of a related protein that on the membrane A. S. G. S. J. H. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). expressed in the protein no a of typical of lysosomal but within the also to be on the membrane To further the sequence for lysosomal in we mutated the and altered the between the and the acidic have been shown to the of the lysosomal protein with and to of the protein S. J. S. J. Biol. 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For to the the acidic lysosomal the cells in a with at marked in the of sialic acid is in cells membrane sialin with cells lysosomal transporter protein, and The activity is with a of which is similar but the for the of sialic acid into lysosomes acid is affected by to a and indicating that the activity is or of these However, is by of lysosomal sialic acid acid and across the lysosomal membrane and several lysosomal transport the sialic acid transport are to this To the of in the activity of the recombinant protein, both the of the and the across the membrane the from to sialin mediated and no transport is the is at the as the In addition, is by at by the with an that mediates the transport activity is affected by of the the that transport is of with that sialin is a sialic acid we to mutations associated with disease the intrinsic transport activity of the protein. To we the of proteins the mutations and This to membrane expression of all of the proteins with the of as by the of and at that the transport activity of the and is no transport activity be for the and The of activity associated with the and mutations and the membrane expression that these mutations transporter However, is at detectable on the with this we the mutation the intrinsic transport activity of the protein. a in transport activity lead to the of a phenotype in of the free sialic acid storage have the normal lysosomal sialic acid transport activity G.M. Beerens C.E. Aula P.P. Verheijen F.W. 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In to the to which mutations trafficking of sialin we found that sialin with and in cells that studies with these be difficult. to mutations trafficking we and proteins expressed in the expression of and sialin to of the by of cells with sialin and a of complete with cells with This is of expression or with the protein is with and the localization is for This with PDI, an endoplasmic protein, in this the of with the also with with sialin with an adaptor protein in a of the biochemical phenotype of Salla disease and infantile sialic acid storage disorder led to the identification of a lysosomal sialic acid transport activity M. Tietze F. Gahl W.A. Science. 1986; 232: 759-762Crossref PubMed Scopus (101) Google Scholar, G.M. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar), and genetic studies suggested that the protein mutated in these mediates this activity (1Verheijen F.W. Verbeek E. Aula N. Beerens C.E. Havelaar A.C. Joosse M. Peltonen L. Aula P. van der Galjaard H. Spek P.J. Mancini G.M. Nat. Genet. 1999; 23: 462-465Crossref PubMed Scopus (218) Google Scholar). Here we a direct for sialin in sialic acid transport and describe the of mutations on the transport activity and trafficking of the protein. The analysis of the sialin sequence from the mutations associated with Salla disease and ISSD occur in within or This degree of is with these a in the function of the protein. Furthermore, the of these with of the protein with a in or of the or in the of the protein within the The derived from a of the is the presence of a in the amino of The an acidic amino acid of a is similar to for between adaptor proteins and their the between and the lysosomal protein 2003; PubMed Scopus Google Scholar, S. J. S. J. Biol. 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The transport activity is by acid, an acidic sugar that is a of the lysosomal sialic acid transport. it in glycoproteins and acid is also a for the transporter and in the lysosomes of individuals with the free sialic acid storage disorders G.M. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, Seppala R. Tietze F. Gahl W.A. J. PubMed Scopus Google Scholar). The DIDS, a transport function R.J. Edwards R.H. Science. 2000; PubMed Scopus Google as as transport. transport systems or be to the and on the lysosomal transport G.M. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google that sialin is directly to the and that there is a between the sugar and However, or sialin a in lysosomal movement of has been suggested for the lysosomal transporter E. M. W. M. Genet. 2000; PubMed Scopus Google Scholar, D.A. S.A. F. Nat. Genet. 1999; PubMed Scopus Google or an has been for the related and A. S. G. S. J. H. F. Proc. Natl. Acad. 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S. A. 1997; PubMed Scopus Google Scholar). the mutation in the related J. and A. transport is by a it is on the the that transport activity movement R.J. Edwards R.H. Science. 2000; PubMed Scopus Google Scholar). Thus, be directly involved in a mechanism for sialin function but function. an mechanism by which mutations lead to a loss of the structure or of the For the with of the protein by the between the predicted that the to In addition, both and are in protein are often at the of and for of Biochim. Biophys. Acta. PubMed Scopus Google Scholar). This that the effect of the and mutations be on the structure of as with the is common to disease-causing mutations in polytopic and A. N. S. J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). in an with of an degradation has been shown to expression and the phenotype of the disease J. C. L. N. E. S. J. Nat. 2002; PubMed Scopus Google Scholar, M. S.A. D. J. S. K. Science. 2004; PubMed Scopus Google Scholar). or a similar effect for the mutation in sialin to be most are the of analysis of the Salla disease mutation (R39C) and the In activity is and protein to with the protein, is to that both mutations are associated with milder forms of the disease (2Aula N. Salomaki P. Timonen R. Verheijen F. Mancini G. Mansson J.E. Aula P. Peltonen L. Am. J. Hum. Genet. 2000; 67: 832-840Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The mutation has been in a single in a with the mutation the clinical phenotype between Salla disease and with ISSD, Salla disease is severe and is associated with of free sialic acid in both and cells from has been the in for Salla disease and ISSD, a complete of sialic acid transport activity in lysosomes derived from with both diseases M. Tietze F. Gahl W.A. Science. 1986; 232: 759-762Crossref PubMed Scopus (101) Google Scholar, 16Tietze F. Seppala R. Renlund M. Hopwood J.J. Harper G.S. Thomas G.H. Gahl W.A. J. Biol. Chem. 1989; 264: 15316-15322Abstract Full Text PDF PubMed Google Scholar, G.M. Beerens C.E. Aula P.P. Verheijen F.W. J. 1991; PubMed Scopus Google Scholar). this has been to a of in the used to transport activity, some has that the Salla mutation activity this and further that there is a direct correlation between clinical phenotype and transporter function. In the function of we have of the mechanisms that Salla disease and We have demonstrated both a direct for the protein in the transport of sialic acid and a loss or severe in transport activity in several The loss of intrinsic function in the transport process for the mutated and further of the effect of similar mutations in the related vesicular glutamate transporters differences these but In addition, the retained activity associated with mutations that therapeutic approaches at the expression or of sialin in these We of the and of the for and