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American Journal of Hematology Oct 2008
Topics: Consanguinity; Humans; Infant; Leukocyte Disorders; Lymphocytes; Male; N-Acetylneuraminic Acid; Sialic Acid Storage Disease
PubMed: 18050357
DOI: 10.1002/ajh.21097 -
Molecular Genetics and Metabolism Dec 2007Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease....
Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease. We have assessed the effect of these mutations and find complete loss of measurable transport activity with both and impaired trafficking of the G409E protein. These results suggest that the two residues are important for proper function of sialin and confirm the association of loss of transport with disease causative mutations.
Topics: Humans; Lysosomes; Mutation, Missense; Protein Transport; Sialic Acid Storage Disease; Symporters
PubMed: 17933575
DOI: 10.1016/j.ymgme.2007.08.121 -
FEBS Letters Sep 2007Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. The key enzyme for the biosynthesis of sialic acid is...
Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the use in biotechnology of sialuria-mutated GNE. Expression of the sialuria-mutated GNE in CHO-cells leads to increased sialylation of recombinant expressed erythropoietin (EPO). Our data show that sialuria-mutated-GNE over-expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as EPO.
Topics: Animals; Bromodeoxyuridine; CHO Cells; Carbohydrate Epimerases; Cell Proliferation; Cricetinae; Cricetulus; Culture Media, Conditioned; DNA; Erythropoietin; Humans; Isoelectric Focusing; Models, Biological; Mutation; N-Acetylneuraminic Acid; Rats; Recombinant Proteins; Sialic Acid Storage Disease
PubMed: 17706199
DOI: 10.1016/j.febslet.2007.07.060 -
Biochimica Et Biophysica Acta Jun 2006Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder...
Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder results in a loss of feedback control of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase by CMP-N-acetylneuraminic acid (CMP-NeuAc) resulting in a substantial overproduction of cytoplasmic free sialic acid. This study addresses fibroblast CMP-NeuAc levels and N- and O-glycan sialylation of serum proteins from Sialuria patients. CMP-NeuAc levels were measured with HPLC in fibroblasts. Isoelectric focusing (IEF) of serum transferrin and of apolipoprotein C-III (apoC-III) was performed on serum of three Sialuria patients. Isoforms of these proteins can be used as specific markers for the biosynthesis of N- and core 1 O-glycans. Furthermore, total N- and O-linked glycans from serum proteins were analyzed by HPLC. HPLC showed a clear overproduction of CMP-NeuAc in fibroblasts of a Sialuria patient. Minor changes were found for serum N-glycans and hypersialylation was found for core 1 O-glycans on serum apoC-III and on total serum O-glycans in Sialuria patients. HPLC showed an increased ratio of disialylated over monosialylated core 1 O-glycans. The hypersialylation of core 1 O-glycans is due to the increase of NeuAcalpha2,6-containing structures (mainly NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc). This may relate to KM differences between GalNAc-alpha2,6-sialyltransferase and alpha2,3-sialyltransferases. This is the first study demonstrating that the genetic defect in Sialuria results in a CMP-NeuAc overproduction. Subsequently, increased amounts of alpha2,6-linked NeuAc were found on serum core 1 O-glycans from Sialuria patients. N-glycosylation of serum proteins seems largely unaffected. Sialuria is the first metabolic disorder presenting with hypersialylated O-glycans.
Topics: Apolipoprotein C-III; Apolipoproteins C; Chromatography, High Pressure Liquid; Glycoproteins; Glycosylation; Humans; Isoelectric Focusing; N-Acetylneuraminic Acid; Nucleotides; Polysaccharides; Protein Isoforms; Sialic Acid Storage Disease; Transferrin
PubMed: 16769205
DOI: 10.1016/j.bbadis.2006.03.009 -
FEBS Letters Sep 2005Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic...
Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.
Topics: Animals; CHO Cells; Cricetinae; Humans; Multienzyme Complexes; N-Acetylneuraminic Acid; Neural Cell Adhesion Molecules; Sialic Acid Storage Disease; Sialyltransferases
PubMed: 16137682
DOI: 10.1016/j.febslet.2005.08.013 -
Journal of Medical Genetics Nov 2005Sialic acid storage diseases (SASDs) are caused by the defective transport of free sialic acid outside the lysosome. Apart from the Salla presentation in Finland, SASD...
BACKGROUND
Sialic acid storage diseases (SASDs) are caused by the defective transport of free sialic acid outside the lysosome. Apart from the Salla presentation in Finland, SASD is a very rare form of lysosomal storage disease (LSD) with approximately 35 cases, all diagnosed after birth, having been reported worldwide. We report a series of 12 French patients with very early manifestations, including eight fetuses diagnosed in utero.
RESULTS
Ultrasound examination, fetal autopsy, or clinical examination showed prominent ascites, rarely progressing to complete hydrops, and highlighted the early severity of bone disease. Dramatic increase of free sialic acid in various biological samples confirmed the diagnosis in all cases. Storage staining affinities and storage distribution in placenta and fetal organs allowed differential diagnosis from other LSDs but cannot differentiate between SASD, sialidosis, and galactosialidosis. Fourteen different mutations were identified, showing the molecular heterogeneity of SASD in the French population. We found that the previously described p.Y306X mutation generated two different transcripts, and we identified seven novel mutations: three deletions (del exon 7, del exons10+11 and c.1296delT), one splice site mutation (c.1350+1G-->T) one nonsense mutation (p.W339X), and two missense mutations (p.R57C and p.G127E).
CONCLUSIONS
The severity of our patients' genotypes is in agreement with their phenotypes but not with the importance and early appearance of the very frequent in utero manifestations. Minimal fetal disease in some patients and a reported case of heterogeneity of fetal involvement within a family suggest that factors other than the genotype influence fetal manifestations.
Topics: Female; Gene Deletion; Genotype; Gestational Age; Humans; Infant; Infant, Newborn; Lysosomal Storage Diseases; Male; Mutation; N-Acetylneuraminic Acid; Phenotype; Pregnancy; Prenatal Diagnosis; Sialic Acid Storage Disease
PubMed: 15805149
DOI: 10.1136/jmg.2004.029744 -
Developmental Medicine and Child... Dec 2004Salla disease, a free sialic acid storage disorder, is one of the 36 currently known disorders in Finland that form the Finnish disease heritage. Salla disease leads to...
Salla disease, a free sialic acid storage disorder, is one of the 36 currently known disorders in Finland that form the Finnish disease heritage. Salla disease leads to learning disability* with a wide clinical variation. Two main categories of the disease have been classified: a conventional subtype and a severe subtype with more severe defects. We present detailed neurocognitive profiles of 41 Finnish patients with Salla disease (19 females, 22 males; age range 11mo to 63y, median 19y). The neurocognitive development of patients with Salla disease was assessed by psychological and neuropsychological testing. All patients were also examined by a paediatric neurologist and a speech therapist. The characteristic cognitive profile consisted of a lower non-verbal performance (mean developmental age 13mo) compared with linguistic skills (mean developmental age 17mo). In particular, spatial and visual-constructive impairments were typical of these patients. Tactile and visual discrimination of forms was poor. Tasks demanding hand-eye coordination, maintenance of visual attention, and those requiring short-term visual memory and executive skills were performed better. Receptive language skills were notably better compared with expressive speech. The patients' interactive and non-verbal communication skills were quite strong. Another typical pattern with Salla disease was severe motor disability. After the second decade of life, the decline in these skills was more pronounced than patients' cognitive deterioration. Our results indicate that even though there is a considerable variation in the clinical findings of patients with Salla disease, the characteristic neurocognitive profile of the disease can be outlined.
Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Cognition Disorders; Dyslexia; Female; Humans; Infant; Language Disorders; Male; Middle Aged; Motor Skills Disorders; Perceptual Disorders; Phenotype; Severity of Illness Index; Sialic Acid Storage Disease; Speech Disorders; Touch; Visual Perception; Wechsler Scales
PubMed: 15581157
DOI: 10.1017/s0012162204001458 -
The Journal of Biological Chemistry Jan 2005Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport...
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.
Topics: Amino Acid Sequence; Biological Transport; Cell Membrane; Conserved Sequence; Endoplasmic Reticulum; HeLa Cells; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Mutation; N-Acetylneuraminic Acid; Organic Anion Transporters; Protein Structure, Tertiary; Protein Transport; Sialic Acid Storage Disease; Symporters
PubMed: 15516337
DOI: 10.1074/jbc.M411295200 -
The EMBO Journal Nov 2004The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid...
The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.
Topics: Amino Acid Motifs; Cell Line; Cell Membrane; Cloning, Molecular; Endocytosis; Humans; Hydrogen-Ion Concentration; Mutation; Organic Anion Transporters; Protein Transport; Sialic Acid Storage Disease; Sialic Acids; Symporters
PubMed: 15510212
DOI: 10.1038/sj.emboj.7600464 -
The Journal of Biological Chemistry Dec 2003Lec3 Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced...
Lec3 Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced polysialic acid (PSA) on neural cell adhesion molecules. To identify the genetic origin of the phenotype, genes encoding different factors required for sialic acid biosynthesis were transfected into Lec3 cells. Only a Gne cDNA encoding UDP-GlcNAc 2-epimerase:ManNAc kinase rescued PSA synthesis. In an in vitro UDP-GlcNAc 2-epimerase assay, Lec3 cells had no detectable UDP-GlcNAc 2-epimerase activity, and Lec3 cells grown in serum-free medium were essentially devoid of sialic acid on glycoproteins. The Lec3 phenotype was rescued by exogenously added N-acetylmannosamine or mannosamine but not by the same concentrations of N-acetylglucosamine, glucosamine, glucose, or mannose. Sequencing of CHO Gne cDNAs identified a nonsense (E35stop) and a missense (G135E) mutation, respectively, in two independent Lec3 mutants. The G135E Lec3 mutant transfected with a rat Gne cDNA had restored in vitro UDP-GlcNAc 2-epimerase activity and cell surface PSA expression. Both Lec3 mutants were similarly rescued with a CHO Gne cDNA and with CHO Gne encoding the known kinase-deficient D413K mutation. However, cDNAs encoding the known epimerase-deficient mutation H132A or the new Lec3 G135E Gne mutation did not rescue the Lec3 phenotype. The G135E Gne missense mutation is a novel mechanism for inactivating UDP-GlcNAc 2-epimerase activity. Lec3 mutants with no UDP-GlcNAc 2-epimerase activity represent sensitive hosts for characterizing disease-causing mutations in the human GNE gene that give rise to sialuria, hereditary inclusion body myopathy, and Nonaka myopathy.
Topics: Amino Acid Sequence; Animals; Blotting, Northern; Blotting, Western; CHO Cells; Carbohydrate Epimerases; Cell Adhesion; Cell Line; Cell Separation; Cricetinae; Culture Media, Serum-Free; DNA, Complementary; Flow Cytometry; Genetic Complementation Test; Glycosylation; Hexosamines; Humans; Lectins; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Mutation, Missense; N-Acetylneuraminic Acid; Phenotype; Plasmids; Point Mutation; Protein Structure, Tertiary; RNA; Rats; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Transfection
PubMed: 14561743
DOI: 10.1074/jbc.M309967200