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PloS One 2012Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in...
Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.
Topics: Amino Acid Sequence; Amino Acids; Animals; Base Sequence; Biocatalysis; Catalytic Domain; Computational Biology; Conserved Sequence; Evolution, Molecular; Exons; Humans; Models, Molecular; Molecular Sequence Data; Multigene Family; Mutagenesis, Insertional; Neuraminidase; Phosphorylation; Phylogeny; Protein Structure, Secondary; Sialyltransferases; Substrate Specificity; Synteny; Vertebrates
PubMed: 22952925
DOI: 10.1371/journal.pone.0044193 -
Proceedings of the National Academy of... Aug 2012In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic...
In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.
Topics: Acids; Adenoviridae; Animals; Anion Transport Proteins; Anions; Biological Transport; Cell Membrane; Fibroblasts; Intracellular Space; Mutation; N-Acetylneuraminic Acid; Nitrate Transporters; Nitrates; Organic Anion Transporters; Protons; Sialic Acid Storage Disease; Submandibular Gland; Sus scrofa; Symporters
PubMed: 22778404
DOI: 10.1073/pnas.1116633109 -
Molecular Therapy : the Journal of the... Jul 2012Recombinant vector systems have been recently identified that when delivered systemically can transduce neurons, glia, and endothelia in the central nervous system...
Recombinant vector systems have been recently identified that when delivered systemically can transduce neurons, glia, and endothelia in the central nervous system (CNS), providing an opportunity to develop therapies for diseases affecting the brain without performing direct intracranial injections. Vector systems based on adeno-associated virus (AAV) include AAV serotype 9 (AAV9) and AAVs that have been re-engineered at the capsid level for CNS tropism. Here, we performed a head-to-head comparison of AAV9 and a capsid modified AAV for their abilities to rescue CNS and peripheral disease in an animal model of lysosomal storage disease (LSD), the mucopolysacharidoses (MPS) VII mouse. While the peptide-modified AAV reversed cognitive deficits, improved storage burden in the brain, and substantially prolonged survival, we were surprised to find that AAV9 provided no CNS benefit. Additional experiments demonstrated that sialic acid, a known inhibitor of AAV9, is elevated in the CNS of MPS VII mice. These studies highlight how disease manifestations can dramatically impact the known tropism of recombinant vectors, and raise awareness to assuming similar transduction profiles between normal and disease models.
Topics: Animals; Brain; Capsid Proteins; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Mice; Mice, Transgenic; Mucopolysaccharidosis VII; N-Acetylneuraminic Acid
PubMed: 22588273
DOI: 10.1038/mt.2012.100 -
Indian Pediatrics Apr 2012Farber disease caused by acid ceramidase deficiency is characterised by a triad of painful and swollen joints, subcutaneous nodules, and laryngeal involvement. A one...
Farber disease caused by acid ceramidase deficiency is characterised by a triad of painful and swollen joints, subcutaneous nodules, and laryngeal involvement. A one year old female with overlapping features of the classical and type 5 variants is reported. Sialuria and elevated plasma chitotriosidase were unusual findings. A novel mutation of the ASAH 1 gene was detected from DNA extracted from the umbilical stump.
Topics: Acid Ceramidase; Farber Lipogranulomatosis; Fatal Outcome; Female; Genotype; Humans; Infant
PubMed: 22565078
DOI: No ID Found -
Journal of Inherited Metabolic Disease Jan 2012
Topics: Female; Fibroblasts; Humans; Pregnancy; Prenatal Diagnosis; Sialic Acid Storage Disease; Tandem Mass Spectrometry
PubMed: 22083207
DOI: 10.1007/s10545-011-9414-5 -
Biochemistry Oct 2011UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE...
UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE isoforms (hGNE1-hGNE3), our database and polymerase chain reaction analysis yielded five additional human isoforms (hGNE4-hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-hGNE8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue N-terminal extension compared to hGNE1. On the basis of similarities to kinases and helicases, this extension does not seem to hinder the epimerase enzymatic active site. hGNE3 and hGNE8 contain a 55-residue N-terminal deletion and a 50-residue N-terminal extension compared to hGNE1. The size and secondary structures of these fragments are similar, and modeling predicted that these modifications do not affect the overall fold compared to that of hGNE1. However, the epimerase enzymatic activity of GNE3 and GNE8 is likely absent, because the deleted fragment contains important substrate binding residues in homologous bacterial epimerases. hGNE5-hGNE8 have a 53-residue deletion, which was assigned a role in substrate (UDP-GlcNAc) binding. Deletion of this fragment likely eliminates epimerase enzymatic activity. Our findings imply that GNE is subject to evolutionary mechanisms to improve cellular functions, without increasing the number of genes. Our expression and modeling data contribute to elucidation of the complex functional and regulatory mechanisms of human GNE and may contribute to further elucidating the pathology and treatment strategies of the human GNE-opathies sialuria and hereditary inclusion body myopathy.
Topics: Amino Acid Sequence; Carbohydrate Epimerases; Catalysis; Catalytic Domain; DNA, Complementary; Gene Deletion; Humans; Models, Molecular; Molecular Sequence Data; N-Acetylneuraminic Acid; Polymerase Chain Reaction; Protein Isoforms; Sequence Homology, Amino Acid; Tissue Distribution
PubMed: 21910480
DOI: 10.1021/bi201050u -
Journal of Neurochemistry Oct 2011Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to...
Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.
Topics: Animals; Aspartic Acid; Baculoviridae; Biological Transport, Active; DNA, Complementary; Glutamic Acid; Humans; Liposomes; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; Organic Anion Transporters; Rats; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sialic Acid Storage Disease; Symporters; Vesicular Biogenic Amine Transport Proteins
PubMed: 21781115
DOI: 10.1111/j.1471-4159.2011.07388.x -
Journal of Inherited Metabolic Disease Oct 2011Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a...
Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution ((13)C(3)-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2-4 h. Reference values in AFS were 0-8.2 μmol/L for 15-25 weeks of gestation and 3.2-12.0 μmol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 μmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20-154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS.
Topics: Amniotic Fluid; Calibration; Cells, Cultured; Chromatography, Liquid; Female; Fibroblasts; Humans; Pregnancy; Pregnancy Trimester, Second; Pregnancy Trimester, Third; Prenatal Diagnosis; Reproducibility of Results; Sialic Acid Storage Disease; Tandem Mass Spectrometry; Urinalysis
PubMed: 21617927
DOI: 10.1007/s10545-011-9351-3 -
Biological & Pharmaceutical Bulletin 2010Aspartate, an excitatory amino acid, is known to be stored in synaptic vesicles and exocytosed from some neurons to perform aspartergic neurotransmission. Through in...
Aspartate, an excitatory amino acid, is known to be stored in synaptic vesicles and exocytosed from some neurons to perform aspartergic neurotransmission. Through in vitro reconstitution, we found that sialin, a lysosomal sialic acid exporter, is responsible for the vesicular storage of aspartate in hippocampal neurons and pinealocytes. Mutations found in Salla disease cause decreased aspartate transport activity without affecting sialic acid transport. Thus, sialin is a multifunctional transporter. It is possible that people with Salla disease lose the ability of aspartergic neurotransmission, and this could explain why Salla disease involves severe neurological defects.
Topics: Animals; Aspartic Acid; Biological Transport; Glutamic Acid; Hippocampus; Humans; Mutation; N-Acetylneuraminic Acid; Organic Anion Transporters; Pineal Gland; Sialic Acid Storage Disease; Synaptic Transmission; Synaptic Vesicles
PubMed: 21048299
DOI: 10.1248/bpb.33.1783 -
The Journal of Biological Chemistry Jun 2010Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled...
Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.
Topics: Amino Acid Sequence; Animals; Cysteine; Glucuronic Acid; Humans; Lysosomal Storage Diseases; Molecular Sequence Data; Mutation; N-Acetylneuraminic Acid; Organic Anion Transporters; Protein Isoforms; Protein Structure, Tertiary; Rats; Sequence Homology, Amino Acid; Structure-Activity Relationship; Symporters
PubMed: 20424173
DOI: 10.1074/jbc.M110.130716