Angel Ashikov

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase *

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.

C. elegans DPY-19 Is a C-Mannosyltransferase Glycosylating Thrombospondin Repeats

Among the different types of protein glycosylation, C-mannosylation of tryptophan residues stands out because of the unique linkage formed between sugar and protein. Instead of the typical O- or N-glycosidic linkage, a C-C bond is used for attachment of a single mannose. C-mannose is characteristically found in thrombospondin type 1 repeats and in the WSXWS motif of type I cytokine receptors. The genetic base of the enzymatic activity catalyzing C-mannosylation was not known. Here we demonstrate that Caenorhabditis elegans DPY-19 is a C-mannosyltransferase. DPY-19 exhibits topological and sequential homology to the N-glycan oligosaccharyltransferase, highlighting an evolutionary link between N- and C-glycosylation. We show that the C. elegans surface receptors MIG-21 and UNC-5 are acceptor substrates of DPY-19 and that C-mannosylation is essential for the secretion of soluble MIG-21. Thereby, our data provide an explanation for the previously described identical Q neuroblast migration phenotypes of dpy-19 and mig-21 mutants.

Molecular Cloning of a Xylosyltransferase That Transfers the Second Xylose to O-Glucosylated Epidermal Growth Factor Repeats of Notch

Background: Notch EGF repeats are glycosylated with xylose containing O-glycans. Results: We have identified a human gene encoding an enzyme transferring the second xylose to generate the Xyl-Xyl-Glc trisaccharide on Notch EGF repeats. Conclusion: Genes encoding all glycosyltransferase activities involved in the O-glucose-linked modification are now known. Significance: Identification of the responsible genes allows elucidation of the biological role of Notch xylosylation. The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseβ1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.

A CMP-sialic acid transporter cloned from Arabidopsis thaliana

Sialylation of glycans is ubiquitous in vertebrates, but was believed to be absent in plants, arthropods, and fungi. However, recently evidence has been provided for the presence of sialic acid in these evolutionary clades. In addition, homologs of mammalian genes involved in sialylation can be found in the genomes of these taxa and for some Drosophila enzymes, involvement in sialic acid metabolism has been documented. In plant genomes, homologs of sialyltransferase genes have been identified, but there activity could not be confirmed. Several mammalian cell lines exist with defects in the sialylation pathway. One of these is the Chinese hamster ovary cell line Lec2, deficient in CMP-sialic acid transport to the Golgi lumen. These mutants provide the possibility to clone genes by functional complementation. Using expression cloning, we have identified an Arabidopsis thaliana nucleotide sugar transporter that is able to complement the CMP-sialic acid transport deficiency of Lec2 cells. The isolated gene (At5g41760) is a member of the triose-phosphate/nucleotide sugar transporter gene family. Recombinant expression of the gene in yeast and testing in vitro confirmed its ability to transport CMP-sialic acid.

NANS-mediated synthesis of sialic acid is required for brain and skeletal development

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.

Structure and function of nucleotide sugar transporters: Current progress

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.

Mutations in SLC35A3 cause autism spectrum disorder, epilepsy and arthrogryposis

Background The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy. Methods and results By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms. Conclusions Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity

Significance Nucleotide sugars are donor substrates for the formation of glycan modifications, which are important for the function of many macromolecules such as proteins and lipids. Although most of the glycosylation reactions occur in the endoplasmic reticulum (ER) and Golgi of eukaryotic cells, nucleotide sugar activation occurs in the cytosol and specific transporters must carry these molecules across the membrane. We identified REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1) as an ER-localized transporter of UDP-GlcNAc and UDP-GalNAc in plants. In contrast to animals, nothing is known about the function of the two respective sugar residues in the plant ER. We demonstrate that ROCK1-mediated transport plays a role in the ER-associated protein quality control and loss of ROCK1 enhances cytokinin responses by suppressing the activity of cytokinin-degrading CKX proteins. The formation of glycoconjugates depends on nucleotide sugars, which serve as donor substrates for glycosyltransferases in the lumen of Golgi vesicles and the endoplasmic reticulum (ER). Import of nucleotide sugars from the cytosol is an important prerequisite for these reactions and is mediated by nucleotide sugar transporters. Here, we report the identification of REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1, At5g65000) as an ER-localized facilitator of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transport in Arabidopsis thaliana. Mutant alleles of ROCK1 suppress phenotypes inferred by a reduced concentration of the plant hormone cytokinin. This suppression is caused by the loss of activity of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKXs). Cytokinin plays an essential role in regulating shoot apical meristem (SAM) activity and shoot architecture. We show that rock1 enhances SAM activity and organ formation rate, demonstrating an important role of ROCK1 in regulating the cytokinin signal in the meristematic cells through modulating activity of CKX proteins. Intriguingly, genetic and molecular analysis indicated that N-glycosylation of CKX1 was not affected by the lack of ROCK1-mediated supply of UDP-GlcNAc. In contrast, we show that CKX1 stability is regulated in a proteasome-dependent manner and that ROCK1 regulates the CKX1 level. The increased unfolded protein response in rock1 plants and suppression of phenotypes caused by the defective brassinosteroid receptor bri1-9 strongly suggest that the ROCK1 activity is an important part of the ER quality control system, which determines the fate of aberrant proteins in the secretory pathway.