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FASEB Journal : Official Publication of... Nov 2008Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused...
Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.
Topics: Alleles; Amino Acid Substitution; Cells, Cultured; Cytidine Monophosphate N-Acetylneuraminic Acid; Fibroblasts; Genes, Dominant; Humans; Multienzyme Complexes; Mutation, Missense; RNA Interference; RNA, Small Interfering; Sialic Acid Storage Disease
PubMed: 18653764
DOI: 10.1096/fj.08-110890 -
Topics in Current Chemistry 2015UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the... (Review)
Review
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the cytosolic formation of CMP-N-acetylneuraminic acid from UDP-N-acetylglucosamine. In this review we give an overview of structure, biochemistry, and genetics of the bifunctional enzyme and its complex regulation. Furthermore, we will focus on diseases related to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
Topics: Animals; Cytidine Monophosphate N-Acetylneuraminic Acid; Disease Models, Animal; Distal Myopathies; Gene Expression Regulation; Genes, Regulator; Humans; Mice; Mice, Transgenic; Multienzyme Complexes; Mutation; Protein Structure, Quaternary; Sialic Acid Storage Disease; Uridine Diphosphate N-Acetylglucosamine
PubMed: 23842869
DOI: 10.1007/128_2013_464 -
The Journal of Biological Chemistry Oct 1989Sialuria is a rare inborn error of metabolism, the hallmarks of which are moderate developmental retardation, coarse facial features, and an enormous amount of free...
Sialuria is a rare inborn error of metabolism, the hallmarks of which are moderate developmental retardation, coarse facial features, and an enormous amount of free N-acetylneuraminic acid (sialic acid) in the urine. Until now, the basic biochemical defect in this disorder has remained uncertain. In this report, the activity of the rate-limiting enzyme in the biosynthesis of sialic acid has been measured directly in whole cell lysates by a highly sensitive assay. With this technique, the basic defect in sialuria has been identified unequivocally as the loss of feedback control of uridine diphosphate N-acetylglucosamine 2-epimerase by cytidine monophosphate N-acetylneuraminic acid with resultant overproduction of sialic acid.
Topics: Carbohydrate Epimerases; Carbohydrate Metabolism, Inborn Errors; Carrier Proteins; Cells, Cultured; Feedback; Fibroblasts; Hexosamines; Humans; Kinetics; N-Acetylneuraminic Acid; Reference Values; Sialic Acids; Skin
PubMed: 2808337
DOI: No ID Found -
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 -
JCI Insight Dec 2018Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid...
Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.
Topics: Adult; Animals; Disease Models, Animal; Edema, Cardiac; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Genetic Diseases, Inborn; HEK293 Cells; Hexosamines; Humans; Male; Muscle, Skeletal; Muscular Diseases; Mutation; N-Acetylneuraminic Acid; Oxo-Acid-Lyases; Sialic Acid Storage Disease; Young Adult; Zebrafish
PubMed: 30568043
DOI: 10.1172/jci.insight.122373 -
ACS Omega Feb 2021Rare diseases impact hundreds of millions of individuals worldwide. However, few therapies exist to treat the rare disease population because financial resources are...
Rare diseases impact hundreds of millions of individuals worldwide. However, few therapies exist to treat the rare disease population because financial resources are limited, the number of patients affected is low, bioactivity data is often nonexistent, and very few animal models exist to support preclinical development efforts. Sialidosis is an ultrarare lysosomal storage disorder in which mutations in the NEU1 gene result in the deficiency of the lysosomal enzyme sialidase-1. This enzyme catalyzes the removal of sialic acid moieties from glycoproteins and glycolipids. Therefore, the defective or deficient protein leads to the buildup of sialylated glycoproteins as well as several characteristic symptoms of sialidosis including visual impairment, ataxia, hepatomegaly, dysostosis multiplex, and developmental delay. In this study, we used a bibliometric tool to generate links between lysosomal storage disease (LSD) targets and existing bioactivity data that could be curated in order to build machine learning models and screen compounds . We focused on sialidase as an example, and we used the data curated from the literature to build a Bayesian model which was then used to score compound libraries and rank these molecules for testing. Two compounds were identified from testing using microscale thermophoresis, namely sulfameter ( 2.15 ± 1.02 μM) and mexenone ( 8.88 ± 4.02 μM), which validated our approach to identifying new molecules binding to this protein, which could represent possible drug candidates that can be evaluated further as potential chaperones for this ultrarare lysosomal disease for which there is currently no treatment. Combining bibliometric and machine learning approaches has the ability to assist in curating small molecule data and model building, respectively, for rare disease drug discovery. This approach also has the capability to identify new compounds that are potential drug candidates.
PubMed: 33553934
DOI: 10.1021/acsomega.0c05591 -
Molecular Therapy. Nucleic Acids Dec 2023Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency...
Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.
PubMed: 37727271
DOI: 10.1016/j.omtn.2023.08.024 -
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 -
Proceedings of the National Academy of... Aug 2008Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes...
Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H(+)/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H(+)/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.
Topics: Adenosine Triphosphate; Amino Acid Transport Systems, Acidic; Animals; Aspartic Acid; Biological Transport; Cells, Cultured; Exocytosis; Glutamic Acid; Hippocampus; Humans; Mice; N-Acetylneuraminic Acid; Neurons; Organic Anion Transporters; Phylogeny; Rats; Rats, Wistar; Symporters
PubMed: 18695252
DOI: 10.1073/pnas.0804015105 -
Science Advances Jan 2023Malfunction of the sialic acid transporter caused by various genetic mutations in the gene encoding Sialin leads to a spectrum of neurodegenerative conditions called...
Malfunction of the sialic acid transporter caused by various genetic mutations in the gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo-electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.
Topics: Humans; N-Acetylneuraminic Acid; Cryoelectron Microscopy; Sialic Acid Storage Disease; Mutation; Symporters; Ion Transport
PubMed: 36662855
DOI: 10.1126/sciadv.ade8346