Glycoprotein Glucosyltransferase Research Articles

Identification of HUGT1 as a potential BiP activator and a cellular target for improvement of recombinant protein production using a cDNA screening system.

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon gamma, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

Involvement of Sep15 family members in immune response

Selenium and selenoproteins are known to have immunomodulatory properties. 15 kDa selenoprotein (Sep15) was previously shown to interact with UDP‐glucose:glycoprotein glucosyltransferase, a protein involved in the calnexin quality control cycle for many secreted glycoproteins, including proteins with the immune protection function (e.g., MHC class I receptor). In this study, we examined the resistance of Sep15 knockout mice (Sep15 KO) to viral infection and autoimmune disease. In both cases the development of the disease in the Sep15 KO group was different from that in wild type mice, and resulted in higher severity of symptoms and higher percentage of death cases. Examination of immunological parameters in mouse spleenocytes, including MHC class I and II, and CD4/CD8 population of T cells, did not show significant differences between WT and Sep15 KO groups. However, cell culture experiments with Sep15 KO MEFs demonstrated a decrease in â2‐microglobulin subunit when Sep15 KO was combined with deficiency in a distant Sep15 homolog, Selenoprotein M. Altogether, these data show the role of Sep15 family members in the immune function.

The Recognition Motif of the Glycoprotein-Folding Sensor Enzyme UDP-Glc:Glycoprotein Glucosyltransferase

The folding of glycoproteins is primarily mediated by a quality control system in the ER, in which UDP-Glc:glycoprotein glucosyltransferase (UGGT) serves as a "folding sensor". In this system, client glycoproteins are delivered to UGGT after the trimming of their innermost glucose residue by glucosidase II, which releases them from the lectin chaperones calnexin (CNX) and calreticulin (CRT). UGGT is inactive against folded proteins, allowing them to proceed to the Golgi apparatus for further processing to complex- or hybrid-type glycoforms. On the other hand, this enzyme efficiently glucosylates incompletely folded glycoproteins to monoglucosylated structures, providing them with an opportunity to interact with CNX/CRT. In order to clarify the mode of this enzyme's substrate recognition, we conducted a structure-activity relationship study using a series of synthetic probes. The inhibitory activities of various glycans suggest that UGGT has a strong affinity for the core pentasaccharide (Man3GlcNAc2) of high-mannose-type glycans. Our comparison of the reactivity of acceptors that have been modified by various aglycons supports the hypothesis that UGGT recognizes the hydrophobic region of client glycoproteins. Moreover, we discovered fluorescently labeled substrates that will be valuable for highly sensitive detection of UGGT activity.

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37 degrees C, it is essential for parasite growth and survival at 40 degrees C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.

Unique Asn-linked Oligosaccharides of the Human Pathogen Entamoeba histolytica

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.

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.

Effects of Macromolecular Crowding on Glycoprotein Processing Enzymes

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.

Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.

The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients

Pancreatic beta cells have highly developed endoplasmic reticulum (ER) due to their role in insulin secretion. Since ER stress has been associated with beta cell dysfunction, we studied several features of beta cell ER in human type 2 diabetes. Pancreatic samples and/or isolated islets from non-diabetic controls (ND) and type 2 diabetes patients were evaluated for insulin secretion, apoptosis (electron microscopy and ELISA), morphometric ER assessment (electron microscopy), and expression of ER stress markers in beta cell prepared by laser capture microdissection and in isolated islets. Insulin release was lower and beta cell apoptosis higher in type 2 diabetes than ND islets. ER density volume was significantly increased in type 2 diabetes beta cells. Expression of alpha-mannosidase (also known as mannosidase, alpha, class 1A, member 1) and UDP-glucose glycoprotein glucosyl transferase like 2 (UGCGL2), assessed by microarray and/or real-time reverse transcriptase polymerase chain reaction (RT-PCR), differed between ND and type 2 diabetes beta cells. Expression of immunoglobulin heavy chain binding protein (BiP, also known as heat shock 70 kDa protein 5 [glucose-regulated protein, 78 kDa] [HSPA5]), X-box binding protein 1 (XBP-1, also known as XBP1) and C/EBP homologous protein (CHOP, also known as damage-inducible transcript 3 [DDIT3]) was not higher in type 2 diabetes beta cell or isolated islets cultured at 5.5 mmol/l glucose (microarray and real-time RT-PCR) than in ND samples. When islets were cultured for 24 h at 11.1 mmol/l glucose, there was induction of BiP and XBP-1 in type 2 diabetes islets but not in ND islets. Beta cell in type 2 diabetes showed modest signs of ER stress when studied in pancreatic samples or isolated islets maintained at physiological glucose concentration. However, exposure to increased glucose levels induced ER stress markers in type 2 diabetes islet cells, which therefore may be more susceptible to ER stress induced by metabolic perturbations.

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an alpha-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing alpha-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-dependent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing alpha-mannosidase-like protein and Mns1 orthologs, respectively, each of which had alpha-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD.

Substrate-Specific Requirements for UGT1-Dependent Release from Calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.

Allele-Specific Suppression of a Defective Brassinosteroid Receptor Reveals a Physiological Role of UGGT in ER Quality Control

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with nonphysiological substrates revealed that UGGT can glucosylate nonnative glycoproteins by recognizing subtle folding defects; however, its physiological function remains undefined. Here, we show that mutations in the Arabidopsis EBS1 gene suppressed the growth defects of a brassinosteroid (BR) receptor mutant, bri1-9, in an allele-specific manner by restoring its BR sensitivity. Using a map-based cloning strategy, we discovered that EBS1 encodes the Arabidopsis homolog of UGGT. We demonstrated that bri1-9 is retained in the ER through interactions with several ER chaperones and that ebs1 mutations significantly reduce the stringency of the retention-based ER quality control, allowing export of the structurally imperfect yet biochemically competent bri1-9 to the cell surface for BR perception. Thus, our discovery provides genetic support for a physiological role of UGGT in high-fidelity ER quality control.

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

N-glycan processing in ER quality control

Glycosylation of asparagine residues in Asn-x-Ser/Thr motifs is a common covalent modification of proteins in the lumen of the endoplasmic reticulum (ER). By substantially contributing to the overall hydrophilicity of the polypeptide, pre-assembled core glycans inhibit possible aggregation caused by the inevitable exposure of hydrophobic patches on the as yet unstructured chains. Thereafter, N-glycans are modified by ER-resident enzymes glucosidase I (GI), glucosidase II (GII), UDP-glucose:glycoprotein glucosyltransferase (UGT) and mannosidase(s) and become functional appendices that determine the fate of the associated polypeptide. Recent work has improved our understanding of how the removal of terminal glucose residues from N-glycans allows newly synthesized proteins to access the calnexin chaperone system; how substrate retention in this specialized chaperone system is regulated by de-/re-glucosylation cycles catalyzed by GII and UGT1; and how acceleration of N-glycan dismantling upon induction of EDEM variants promotes ER-associated degradation (ERAD) under conditions of ER stress. In particular, characterization of cells lacking certain ER chaperones has revealed important new information on the mechanisms regulating protein folding and quality control. Tight regulation of N-glycan modifications is crucial to maintain protein quality control, to ensure the synthesis of functional polypeptides and to avoid constipation of the ER with folding-defective polypeptides.

AtUTr1, a UDP-glucose/UDP-galactose Transporter from Arabidopsis thaliana, Is Located in the Endoplasmic Reticulum and Up-regulated by the Unfolded Protein Response

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.

Characterization of Sep15, a novel thioredoxin‐like selenoprotein, and its interaction with UDP‐glucose:glycoprotein glucosyltransferase

Selenium is an important trace element with potent cancer prevention activity. The 15-kDa selenoprotein (Sep15) has been proposed to mediate the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, UDP-glucose:glycoprotein glucosyltransferase (GT), an endoplasmic reticulum chaperone involved in assessing fidelity of protein folding, was identified as the binding partner of Sep15. In this study we used co-immunoprecipitation and isothermal titration calorimetry to characterize interactions between members of GT and Sep15 protein families. Sep15 and GT formed a tight 1:1 complex, and this interaction was mediated by a novel cysteine-rich domain located at the N-terminus of Sep15. We also measured redox potential of Sep15, which is similar to that of protein disulfide isomerase suggesting that the C-terminal domain that contains a redox active CxU motif may be involved in the formation and isomerization of disulfide bonds. In mammals, Sep15 expression was regulated by dietary selenium, and either increased expression or depletion of Sep15 by RNA interference altered redox homeostasis. Taken together, these observations suggest that Sep15 may link cancer preventive effects of selenium and the quality control machinery of the endoplasmic reticulum. This work was supported by a NIH grant CA080946.

Mechanisms of Pharmacological Rescue of Trafficking-defective hERG Mutant Channels in Human Long QT Syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Exploration of oligosaccharide‐protein interactions in glycoprotein quality control by synthetic approaches

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides, play important roles in glycoprotein quality control. This process is highly complex, involving a number of lectins, chaperones, and glycan-processing enzymes. For example, calnexin and calreticulin (CRT) are molecular chaperones that recognize monoglucosylated forms of high-mannose-type glycans. UDP-glucose : glycoprotein glucosyltransferase (UGGT) only glucosylates high-mannose-type glycans attached to partially folded proteins. Fbs1 is a component of ubiquitin ligase that recognizes sugar chains. Although recent studies have clarified the properties of these proteins, most of them used oligosaccharides derived from natural sources, which contain structural heterogeneity. In order to gain a more precise understanding, we started our program to comprehensively synthesize high-mannose-type glycans associated with a protein quality control system. Additionally, investigation of artificial glycoproteins led us to the discovery of the first nonpeptidic substrate of UGGT. These synthetic oligosaccharide probes have allowed us to conduct quantitative evaluations of the activity and specificity of CRT, Fbs1, and UGGT.

Synthetic Substrates for an Endoplasmic Reticulum Protein‐Folding Sensor, UDP‐Glucose: Glycoprotein Glucosyltransferase

Quality control: UDP-glucose:glycoprotein glucosyltransferase (UGGT) works as the folding sensor in glycoprotein quality control. It glucosylates the Man9GlcNAc2 of misfolded glycoproteins to produce Glc1Man9GlcNAc2, which is a ligand of calnexin and calreticulin. The synthetic substrate Man9GlcNAc2-MTX can be used for the quantitative analysis of UGGT. UDP=uridine 5′-diphosphate, Glc=D-glucose, Man=D-mannose, GlcNAc=N-acetyl-D-glucosamine, MTX=methotrexate.

A Novel Cysteine-rich Domain of Sep15 Mediates the Interaction with UDP-glucose:Glycoprotein Glucosyltransferase

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.