Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter. Blood

Inistitut National de la Santé et de la Recherche Médicale (INSERM U504), Groupement de Recherche (GDR), Centre National de la Recherche Scientifque (CNRS) 2590, University of Paris Sud XI, Villejuif 94807, France.
Blood (Impact Factor: 10.45). 05/2005; 105(7):2671-6. DOI: 10.1182/blood-2004-09-3509
Source: PubMed


We have identified a homozygous G>A substitution in the donor splice site of intron 6 (IVS6 + 1G>A) of the cytidine monophosphate (CMP)-sialic acid transporter gene of Lec2 cells as the mutation responsible for their asialo phenotype. These cells were used in complementation studies to test the activity of the 2 CMP-sialic acid transporter cDNA alleles of a patient devoid of sialyl-Le(x) expression on polymorphonuclear cells. No complementation was obtained with either of the 2 patient alleles, whereas full restoration of the sialylated phenotype was obtained in the Lec2 cells transfected with the corresponding human wild-type transcript. The inactivation of one patient allele by a double microdeletion inducing a premature stop codon at position 327 and a splice mutation of the other allele inducing a 130-base pair (bp) deletion and a premature stop codon at position 684 are proposed to be the causal defects of this disease. A 4-base insertion in intron 6 was found in the mother and is proposed to be responsible for the splice mutation. We conclude that this defect is a new type of congenital disorder of glycosylation (CDG) of type IIf affecting the transport of CMP-sialic acid into the Golgi apparatus.

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    • "which is capable of transporting UDPglucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) [45]. Multi-substrate specificity may be partially explained by a common evolutionary ancestor [38], or alternatively recent studies have proposed that NST redundancy may be an evolutionary backup mechanism in case of NST impairment, deletion or mutation [15]. Fig. 1. "
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    ABSTRACT: The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure–function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.
    Computational and Structural Biotechnology Journal 06/2014; 10(16). DOI:10.1016/j.csbj.2014.05.003
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    • "Thus it is possible that the p.Ser213Phe mutation causes loss of function in the UGT, though further functional studies are required for clarifying pathogenicity of this missense allele. Mutations in three nucleotide-sugar transporters have been previously reported to cause defects in glycan biosynthesis in humans: the GDP-fucose transporter encoded by SLC35C1, the CMP-sialic acid transporter encoded by SLC35A1, and the UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter encoded by SLC35D1 [Hiraoka et al., 2007; Liu et al., 2010; Lubke et al., 2001; Luhn et al., 2001; Martinez-Duncker et al., 2005]. Similarly, a defect in UGT would cause UDP-galactose deficiency in the ER–Golgi network, leading to reduced galactosylation of glycoproteins, glycosphingolipids , and proteoglycans [Liu et al., 2010]. "
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    ABSTRACT: Early onset epileptic encephalopathies (EOEE) are severe neurological disorders characterized by frequent seizures accompanied by developmental regression or retardation. Whole-exome sequencing of 12 patients together with five pairs of parents and subsequent Sanger sequencing in additional 328 EOEE patients identified two de novo frameshift and one missense mutations in SLC35A2 at Xp11.23, respectively. The three patients are all females. X-inactivation analysis of blood leukocyte DNA and mRNA analysis using lymphoblastoid cells derived from two patients with a frameshift mutation indicated that only the wild-type SLC35A2 allele was expressed in these cell types, at least in part likely as a consequence of skewed X-inactivation. SLC35A2 encodes a UDP-galactose transporter, which selectively supplies UDP-galactose from the cytosol to the Golgi lumen. Transient expression experiments revealed that the missense mutant protein was correctly localized in the Golgi apparatus. In contrast, the two frameshift mutant proteins were not properly expressed, suggesting that their function is severely impaired. Defects in the UDP-galactose transporter can cause congenital disorders of glycosylation. Of note, no abnormalities of glycosylation were observed in three serum glycoproteins, which is consistent with favorably skewed X-inactivation. We hypothesize that a substantial number of neurons might express the mutant SLC35A2 allele and suffer from defective galactosylation, resulting in EOEE. This article is protected by copyright. All rights reserved.
    Human Mutation 12/2013; 34(12). DOI:10.1002/humu.22446 · 5.14 Impact Factor
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    • "The Pro5 cell line allows normal protein sialylation, while the Lec2 cell line produces essentially non-sialylated proteins due to a deficiency in the CMP-sialic acid transporter [23] [24]. The Lec2 cell line used in this study serves as a model for CDG type IIf [25] [26]. "
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    ABSTRACT: Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K(+) (K(v)) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K(v) channel isoform, K(v)1.5, but have no effect on gating of a second Shaker isoform, K(v)1.4. Each isoform contains one potential N-glycosylation site located along the S1-S2 linker; immunoblot analyses verified that K(v)1.4 and K(v)1.5 were N-glycosylated. The conductance-voltage (G-V) relationships and channel activation rates for two glycosylation-site deficient K(v)1.5 mutants, K(v)1.5(N290Q) and K(v)1.5(S292A), and for wild-type K(v)1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing approximately 18 mV compared to wild-type K(v)1.5 activation. External divalent cation screening experiments suggested that K(v)1.5 sialic acids contribute to an external surface potential that modulates K(v)1.5 activation. Channel availability was unaffected by changes in K(v)1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K(v)1.5 activation. The sialic acids fully account for effects of N-glycans on K(v)1.5 gating. Conversely, K(v)1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K(v)1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K(v)1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.
    Biochimica et Biophysica Acta 12/2009; 1798(3):367-75. DOI:10.1016/j.bbamem.2009.11.018 · 4.66 Impact Factor
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