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At least 19 recordsLinked to original sources
PubMed1996

Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator.

We have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1,179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described.

Amino Acid Sequence↗
PubMed1993

The UDP-galactose translocator gene is mapped to band Xp11.23-p11.22 containing the Wiskott-Aldrich syndrome locus.

We have cloned a segment of the human gene encoding UDP-galactose translocator by genetic complementation of its defective mutant in mouse FM3A cells. Chromosome mapping using fluorescent in situ hybridization revealed that the cloned gene hybridized to the Xp11.23-11.23 region of the X chromosome. This region is shared by the locus of Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency disorder, characterized by defective sugar chains on cell surface components. Genetic and phenotypic similarities suggest a possible link between UDP-galactose translocator and the Wiskott-Aldrich syndrome (WAS).

Animals↗
PubMed1988

A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex.

Two lectin-resistant mutants derived from a polarized epithelial cell line have been described (Meiss, H.K., Green, R.F., and Rodriguez-Boulan, E.J. (1982) Mol. Cell. Biol. 2, 1287-1294). One of these mutants, the Madin-Darby canine kidney strain II cell line resistant to Ricinus communis agglutinin (MDCKII-RCAr), has been further characterized, and the biochemical defect leading to its altered phenotype has been determined. MDCKII-RCAr cells are shown to be enriched in cell-surface glycoconjugates bearing terminal N-acetylglucosamine residues by in vitro exogalactosylation and by labeling with fluorescent lectins. Binding assays with a sialic acid-specific lectin reveal a 70-75% reduction in sialylation of cell-surface glycoconjugates. The defect is pleiotropic in nature, affecting glycoproteins as well as glycosphingolipids. Analysis of glycosphingolipids shows a strong reduction of galactose-containing glycosphingolipids. Almost 90% of the glycosphingolipids are identified as glucosyl-ceramide. The mutant is not deficient in galactosyl- and sialytransferase activities. However, Golgi vesicles isolated from MDCKII-RCAr cells translocate UDP-galactose at only 2% of the rate observed for vesicles from wild-type MDCKII cells. The deficiency is specific, because translocation rates of UDP-N-acetylglucosamine and CMP-sialic acid are comparable for vesicles isolated from MDCKII-RCAr cells and wild-type cells. Despite the inability to translocate UDP-galactose into the lumen of the Golgi apparatus, MDCKII-RCAr cells are able to form monolayers with normal apical and basolateral polarity as shown by plasma membrane domain-restricted exogalactosylation.

Animals↗
PubMed1986

Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes.

The biochemical defect in the mutant Chinese hamster ovary cell lines Clone 13 (Briles, E. B., Li, E., and Kornfeld, S. (1977). J. Biol. Chem. 252, 1107-1116) and Lec8 (Stanley, P. (1980) ACS Symp. Ser. 128, 214-221) was examined. These two mutants, which belong to the same genetic complementation group, were shown in previous studies to exhibit an 80-90% reduction in galactosylation and sialylation of proteins and lipids when compared to wild-type cells. The same studies, however, demonstrated that the mutants were not deficient in the corresponding sugar nucleotides, glycosyltransferases, and endogenous acceptors for these transferases. We now provide evidence strongly suggesting that the primary defect in Lec8 and Clone 13 cells is their inability to translocate UDP-galactose into the lumen of the Golgi apparatus. Golgi vesicles from Lec8 and Clone 13 CHO glycosylation mutants translocate in vitro UDP-galactose at only 3-5% the rate of vesicles from wild-type CHO cells. The deficiency is specific because vesicles from the mutant cells can translocate adenosine 3'-phosphate 5'-phosphosulfate, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine at rates comparable to those of vesicles from wild-type cells. These studies also suggest that sugar nucleotides sharing a common uridine nucleotide utilize different translocators present in the Golgi membrane in vivo. The consequence of the above-described mutations and the resulting block in galactosylation of macromolecules in vivo on the translocation of CMP-sialic acid into the Golgi lumen was also examined. As expected, Golgi apparatus vesicles from Lec8 cells were unable to incorporate sialic acid into (endogenous) macromolecules. However, the vesicles were able to transport CMP-sialic acid into their lumen, although the rate of translocation was only 17% of that of wild-type-derived Golgi vesicles.

Animals↗
PubMed1985

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus.

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.

Animals↗
PubMed1993

5-Propyl-2-deoxyuridine induced interference with glycosylation in herpes simplex virus infected cells. Nature of PdU-induced modifications of N-linked glycans.

In herpes simplex virus-infected (HSV) cells, the antiviral nucleoside analogue 5-n-propyl-2'-deoxyuridine (PdU) may, under certain circumstances, induce a pattern of interference with late steps in formation of N-linked glycans, resulting in increased availability of viral glycoproteins for neutralizing antibodies. The PdU-induced changes in N-linked glycans, released by pronase digestion of the HSV-specified glycoprotein gC-1, were investigated by using lectin affinity chromatography and Bio-Gel P6 gel filtration of glycans, radiolabelled with [3H]galactose or [3H]glucosamine. PdU-treatment of HSV-infected cells totally inhibited addition of sialic acid and reduced the amount of galactose incorporated into N-linked glycans by 70%. In addition, the PDU-treatment caused a decrease in oligosaccharides with affinity for Phaseoulus vulgaris leuco-agglutinin and erythro-agglutinin, and an increase in Lens culinaris lectin (LCA)-binding oligosaccharides, suggesting a PdU-induced shift from multi-branched to moderately branched structures. This shift was also found in HSV-infected B16 mouse melanoma cells, where the large content of multi-branched oligosaccharides contributes to the metastatic potential. The LCA-binding glycans from PdU-treated cells were smaller and contained less galactose units than corresponding structures from untreated cells. In a cell-free system, PdU 5'-monophosphate inhibited the translocation of UDP-GlcNAc, and, to a smaller extent, also the translocation of UDP-galactose into Golgi vesicles, suggesting that nucleotide sugar translocation is one important target for the PdU-induced interference with glycosylation in HSV-infected cells.

Agglutinins↗
PubMed1997

The Schizosaccharomyces pombe gms1+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation.

In a previous study, we isolated a Schizosaccharomyces pombe mutant defective in protein galactosylation (Takegawa, K., Tanaka, N., Tabuchi, M. and Iwahara, S. (1996) Biosci. Biochem. Biotech. 60, 1156-1159). From an S. pombe genomic library, we cloned the gms1+ gene which restored the galactosylation of cell wall glycoproteins. Gms1 protein shares significant sequence similarity with human UDP-galactose and murine CMP-sialic acid transporters. The fission yeast strains deleted for the gms1+ gene lacked galactose residues in sell surface glycoproteins and were significantly decreased in UDP-galactose transport activity. These results showed that the gms1+ encodes an UDP-galactose transporter, and this protein appears to be an essential role for the incorporation of UDP-galactose into the lumen of Golgi in s. pombe.

Amino Acid Sequence↗
PubMed1997

Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.

The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.

Animals↗
PubMed1998

Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay.

The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S. cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J. F. (1996) J. Biol. Chem. 271, 3398-3405). However, this is quite unexpected, because S. cerevisiae does not have galactose residues in its glycoproteins. To address this question we have constructed S. cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter in S. cerevisiae. (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium. (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay. This transport in S. cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100. The overall UDP-galactose transport in S. cerevisiae is comparable with that in S. pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S. pombe than in S. cerevisiae.

Biological Transport↗
PubMed1996

Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family.

We described recently the molecular cloning of human UDP-galactose transporter 1 (hUGT1) [Miura, N. et al. (1996) J. Biochem. 120, 236-241]. Now we have characterized its isoform, hUGT2, that is most likely generated through the alternative splicing of a transcript derived from the UGT genomic gene, that also codes for hUGT1. Introduction of the open reading frame sequence of hUGT2 into a mouse cell line, Had-1, that lacks the UDP-galactose transporter, complemented the genetic defect of the mutant, as judged from the lectin-sensitivity spectra of the transformants and the nucleotide-sugar transporting activity of microsomal vesicles isolated from them. UGT-related genes were found through a BLAST search of dbEST based on their significant similarity with hUGT genes. We report here cDNA clones belonging to two subfamilies of the nucleotide-sugar transporter gene family. One is the human CMP-sialic acid transporter gene, and the other is a group of homologous genes with an undefined function that are distributed in man, mouse, and rat, and show significant similarity to the yeast UDP-N-acetylglucosamine transporter.

Amino Acid Sequence↗
PubMed1997

Expression of the human UDP-galactose transporter in the Golgi membranes of murine Had-1 cells that lack the endogenous transporter.

In our previous study, we demonstrated that UDP-galactose transporter cDNAs (hUGT1 and hUGT2) were able to complement the genetic defect of murine Had-1 cells that were deficient in the UDP-galactose transporter, and that the microsomal vesicles isolated from Had-1-transformants, which were obtained through transfection with these cDNAs, had recovered the ability to uptake UDP-galactose [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. In this report, we describe the preparation of peptide antibodies that recognize the hUGT isozymes, and the detection of hUGT proteins expressed in the transformants. The occurrence of the endogenous hUGT1 protein in HeLa cells was also detected. Using the hUGT1-specific antibodies, the subcellular localization of hUGT1 in the Golgi membrane was demonstrated by immunofluorescence microscopy and subcellular fractionation. These studies led us to develop a simple procedure, based on Percoll density gradient centrifugation, for preparing functional Golgi vesicles from the hUGT1-transformed Had-1 cells, that will facilitate future biochemical analyses of the UDP-galactose transporter for the elucidation of its structure-function relationship.

Amino Acid Sequence↗
PubMed1998

Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies.

Nucleotide sugar transporters are mainly located in the Golgi membranes and carry nucleotide sugars, that are produced outside the Golgi apparatus, into the organelle, where they serve as substrates for the elongation of carbohydrate chains by glycosyltransferases. They are thus indispensable for cellular glycoconjugate synthesis and, moreover, may have regulatory roles in producing the structural variety of cellular glycoconjugates. Their occurrence has long been well recognized, but studies on the molecular bases of their strict substrate specificities and modes of action have been hampered by the lack of information on their precise molecular structures. Complementary DNAs encoding several of these transporters were cloned recently, which represented a substantial step forward as to the above mentioned issues. The products of these cDNAs are mutually related hydrophobic proteins consisting of 320-400 amino acid residues with multiple putative transmembrane helix domains, and are located in the Golgi apparatus. This review briefly summarizes the present status of the field of nucleotide sugar transporter research, and also presents an outlook of the study in this field.

Amino Acid Sequence↗
PubMed1998

Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae.

We describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae. Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter. The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus. The hUGT product was expressed in the membranes of Golgi and other organellar compartments. The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity. The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 microM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles. Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles. Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins. The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters.

Biological Transport↗
PubMed1986

Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum.

We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.

Animals↗
PubMed1987

The role of asparagine-linked carbohydrate in natural killer cell-mediated cytolysis.

Chinese hamster ovary cell lines with specific lesions in the formation of glycoconjugates were tested for their sensitivity to lysis by interferon-boosted human natural killer cells. We report here that the type of asparagine-linked carbohydrate present on target cell glycoproteins determines their susceptibility to natural killer lysis. The targets tested were Chinese hamster ovary parent cells and Lec1, Lec2, and Lec8 mutants. Lec8 and Lec2 cells show an overall reduction of galactose and/or sialic acid in their glycoconjugates due to defects in the translocation of UDP-galactose and CMP-sialic acid, respectively. Due to a specific block in N-linked carbohydrate processing, Lec1 cells produce only high mannose-type oligosaccharides, but their glycolipids are identical to those of the parent. Both Lec2 and Lec8 mutants are more sensitive to natural killer lysis than the parent cells. This is consistent with their extensive reduction in cell surface sialic acid. Furthermore, Lec1 mutants are more susceptible to natural killer lysis than the parent cells. To confirm that the increased natural killer sensitivity of Lec1 cells was due to the modification of N-linked carbohydrate, parent cells were treated with swainsonine, a specific inhibitor of N-linked oligosaccharide processing. Swainsonine-treated parent cells are nearly as sensitive to natural killer lysis as the Lec1 mutants.

Alkaloids↗
PubMed1985

Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose.

UDP-galactose appears to be produced on one side of a membrane barrier, opposite the galactosyltransferases that use it as a sugar donor. The translocation of activated galactose across membranes was studied in rat submaxillary-gland microsomal vesicles and in rat liver Golgi vesicles. When these intact vesicles containing the acceptor, N-acetylglucosamine, were incubated in the presence of UDP-galactose and two inhibitors of galactosyltransferase activity, the product, N-acetyl-lactosamine, formed within the vesicles. Thus at least the galactose moiety of UDP-galactose crossed the membranes. When intact Golgi vesicles were incubated with UDP-galactose labelled in both the uridine and the galactose moieties, labelled N-acetyllactosamine was again produced in the vesicles, but less than stoichiometric amounts of the uridine label was found there. Calculation of internal and external concentrations of UMP, a major product released from the cleaved uridine moiety, showed that the vesicles were actually enriched in UMP. When free UMP was incubated with the vesicles, this enrichment did not occur. This result was direct evidence for facilitated transport of UDP-galactose into the Golgi for use by galactosyltransferase.

Amino Sugars↗
PubMed1997

Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium.

Animals↗