A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.     

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.     

Glycoprotein reglucosylation

Proteins following the secretory pathway acquire their proper tertiary and in certain cases also quaternary structures in the endoplasmic reticulum (ER). Incompletely folded species are retained in the ER and eventually degraded. One of the molecular mechanisms by which cells achieve this conformational sorting is based on monoglucosylated N-glycans (Glc1Man5-9GlcNAc2) present on nascent glycoproteins in the ER. This chapter discusses two of the steps that regulate the abundance of such N-glycan structures, including glycoprotein deglucosylation (by glucosidase II) and reglucosylation (by the UDP-Glc:glycoprotein glucosyltransferase), as well as an overview of methods to evaluate the N-glycans prevalent during glycoprotein biogenesis in the ER.     

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.     

The ER UDPase ENTPD5 promotes protein N-glycosylation, the Warburg effect, and proliferation in the PTEN pathway

PI3K and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinases that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. We report here that ENTPD5, an endoplasmic reticulum (ER) enzyme, is upregulated in cell lines and primary human tumor samples with active AKT. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN null cells causes ER stress and loss of growth factor receptors. ENTPD5, together with cytidine monophosphate kinase-1 and adenylate kinase-1, constitute an ATP hydrolysis cycle that converts ATP to AMP, resulting in a compensatory increase in aerobic glycolysis known as the Warburg effect. The growth of PTEN null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an integral part of the PI3K/PTEN regulatory loop and a potential target for anticancer therapy.     

Purification and characterization of a guanosine diphosphatase activity from calf liver microsomal salt wash proteins

A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleoside diphosphates and triphosphates tested, only GDP and UDP are hydrolyzed by the enzyme. The relative affinity of the enzyme for GDP is, however, much higher than for UDP. The effect of the enzyme on the binary complex formed between eukaryotic initiation factor 2 (eIF-2) and GDP has also been investigated. The enzyme neither hydrolyzes GDP bound to eIF-2 nor catalyzes the exchange of eIF-2-bound GDP with GTP even in the presence of Met-tRNAf. The enzyme, therefore, is presumably not involved in recycling of eIF-2 in eukaryotic polypeptide chain initiation reaction. The possible biological function of the enzyme in maintaining the cellular pool of GTP-GDP is discussed.     

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [¹⁴C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[¹⁴C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.     

The role of nucleoside diphosphatase in a uridine nucleotide cycle associated with lactose synthesis in rat mammary-gland Golgi apparatus.

1. UDP-galactose utilization by isolated Golgi vesicles or rat mammary gland synthesizing lactose causes accumulation of UMP but not UDP, although UDP is the immediate product of lactose synthase (EC 2.4.1.22). 2. This can be ascribed to a nucleoside diphosphatase (EC 3.6.1.6), specific for UDP, GDP and IDP, activated by bivalent metal ions and apparently located on the luminal face of the Golgi membrane. 3. The uridine diphosphatase activity exceeds the total galactosyltransferase activity 5-fold, and is estimated to maintain UDP at about 14 micrometer within the Golgi lumen. 4. Evidence is given that UMP, but not UDP, penetrates the membrane and that UMP is rephosphorylated to UDP by a UMP kinase located in the cytosol. 5. Golgi-cytosol relationships with respect to lactose synthesis are formulated in terms of a uridine nucleotide cycle which throws new light on the energy cost and possible regulation of lactose synthesis.     

Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I alpha-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.     

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.     

NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.     

Absence of nucleoside diphosphatase activities in the yeast secretory pathway does not abolish nucleotide sugar-dependent protein glycosylation

It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.     

Nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase

It was found that nucleoside 5'-diphosphates could serve as effectors of ribonucleotide reductase. ADP was an activator of CDP reduction; ADP reduction was activated by dGDP; GDP reduction was activated by dTDP. Conversely, dADP inhibited the reduction of CDP, UDP, GDP, and ADP; dGDP inhibited UDP and GDP reductions; and dTDP inhibited UDP reduction. The inhibition of UDP reduction by dADP, dTDP, and dGDP was at least equal to that observed for dATP, dTTP, and dGTP, respectively. In these experiments with the nucleoside diphosphates as effectors, high-pressure liquid chromatography analysis of the reaction mixtures showed that no nucleoside 5'-triphosphates were found during the reaction period which could account for the effects seen with the nucleoside diphosphates as effectors. Further experiments were carried out in which adenyl-5'-yl imidodiphosphate was used as the positive effector of CDP and UDP reductions in place of ATP. Under these conditions, CDP and UDP reductions were inhibited by dADP, dTDP, and dGDP to the same extent observed in the presence of ATP. ADP served not only as a substrate for ribonucleotide reductase but also as an activator of CDP and UDP reductions. The direct products (dNDPs) also served as positive and negative effectors. Dixon plots indicated that the dNDPs were acting as noncompetitive inhibitors with respect to the substrate. ADP increased the sedimentation velocity of the ribonucleotide reductase in a manner similar to ATP. These data are consistent with the allosteric effects seen with the nucleoside 5'-triphosphates. Additionally, from the thorough study of the role of effectors on UDP reduction, it is clear that UDP reduction was most sensitive to the negative effectors dATP, dADP, dTTP, dTDP, dGTP, and dGDP.     

Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.     

Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation

We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four alpha1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man(5-6)GlcNAc(2) and for ERAD in cells in vivo, leading to the accumulation of Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2) on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.     

The single NTPase gene of Drosophila melanogaster encodes an intracellular nucleoside triphosphate diphosphohydrolase 6 (NTPDase6)

I report here the cloning and characterization of a nucleoside triphosphate diphosphohydrolase 6 (NTPDase6) encoded by the single Dmel/NTPase gene of Drosophila melanogaster. S2 cells stably transfected with the Drosophila NTPDase6 cDNA displayed strong UDPase activity only after addition of NP-40, indicating the intracellular location of the enzyme. The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. It was not or only partially inhibited by several modulators of the cell surface NTPDases, but was strongly inhibited upon oxidative cross-linking by copper phenanthroline. The decrease of activity correlated with dimer formation. Mutagenesis studies indicated that dimer formation required C42 in the transmembrane domain and C447 in the exoplasmic domain. Fluorescence microscopy revealed that the protein was located primarily in the ER. The substrate specificity and cellular localization of the Drosophila NTPDase6 suggest that it participates in Drosophila glycoprotein processing.     

A 31P-NMR study of the interaction of Mg2+ ions with nucleoside diphosphates.

The interaction of Mg2+ with nucleoside disphosphates : ADP, GDP, CDP and UDP has been studied by phosphorus magnetic resonance spectroscopy in aqueous solution. The results show that these four nucleotides behave similarly, the Mg2+ ion binds to the alpha but not to the beta phosphate moiety. The strength of the interaction of Mg2+ ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. The association of Mg2+ on the phosphate chain is stronger in a neutral than in an acid medium.     

Uridine diphosphate-glucose transport into the endoplasmic reticulum of Saccharomyces cerevisiae: in vivo and in vitro evidence

It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP-glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER-containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP-glucose was temperature dependent and saturable with an apparent Km of 46 microM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP-N-acetylglucosamine did not enter these vesicles. Demonstration of UDP-glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP-glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Delta, lack cell wall beta-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Delta mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.