Glycoprotein Folding Research Articles

Single-cell analysis identified PDIA3 as regulator of malignant characteristics and macrophage function in human cancers.

Protein disulfide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER) protein. It has different functions including glycoprotein folding in the ER. The unfavorable prognosis of cancer patients was related to the abnormal PDIA3 expression level. However, it is unclear how PDIA3 correlates with the malignant characteristics of different tumors and its impact on tumor immunity. Pan-cancer data were downloaded from several databases for large-scale bioinformatics analysis. The immunological functions of PDIA3 were systematically explored at the single-cell sequencing level, including cell communication, cell metabolism, cell evolution and epigenetic modification. We performed immunofluorescence staining to visualize PDIA3 expression and infiltration of macrophages in pan-cancer samples. Further, we performed a loss-of-function assay of PDIA3 in vitro. The CCK8 assay, clone formation assay, and transwell assay were performed. M2 macrophages were co-cultured with different cell lines before the transwell assay was performed. The immunofluorescence staining of pan-cancer samples presented a higher expression of PDIA3 than those of the paired normal tissues. According to single-cell sequencing analysis, expression of PDIA3 was closely associated with cell communication, cell metabolism, cell evolution and epigenetic modification. The knockdown of PDIA3 in tumor cells inhibited cell proliferation and invasion, and restrained cocultured M2 macrophage migration. Furthermore, PDIA3 displayed predictive value in immunotherapy response in human cancer cohorts, indicating a potential therapeutic target. Our study showed that PDIA3 was associated with tumor malignant characteristics and could mediate the migration of M2 macrophages in various tumor types. PDIA3 could be a promising target to achieve tumor control and improve the immune response on a pan-cancer scale.

Optimizing Pichia pastoris protein secretion: Role of N-linked glycosylation on the α-mating factor secretion signal leader

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIβ knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by β1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47μM binding affinity for CtUGGTGT24invitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an invitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin.

Dengue virus (DENV) is a major human pathogen that can cause hemorrhagic fever and shock syndrome. One important factor of DENV pathogenicity is non-structural protein 1 (NS1), a glycoprotein that is secreted from infected cells. Here we study the mode of action of the widely used drug ivermectin, used to treat parasitic infections and recently shown to lower NS1 blood levels in DENV-infected patients. We found that ivermectin blocks the nuclear transport of transcription factors required for the expression of chaperones that support the folding and secretion of glycoproteins, including NS1. Impairing nuclear transport of these transcription factors by ivermectin or depleting them from infected cells dampens NS1 folding and thus its secretion. These results reveal a novel mode of action of ivermectin that might apply to other flaviviruses as well.

A Tribute to Richard Lerner (1938–2021) who Encouraged a Glycobiology Approach to a Broad‐Spectrum Antiviral

AbstractRichard Lerner was a visionary polymath of the 20th century; a medical doctor, chemist, immunologist, inventor, and founder of new scientific fields. As a tribute to his life and scientific achievements, we discuss his connection with Oxford University and the Glycobiology Institute and we discuss our research data on the host glycoprotein folding pathway and how its inhibition can lead to broad spectrum antiviral drugs. This host targeting approach for developing antiviral therapies has the advantage of being effective against many viruses, therefore can be used to prevent new epidemics to become pandemics.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint.

None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP-glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals-with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants.

Severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) can infect various human organs, including the respiratory, circulatory, nervous, and gastrointestinal ones. The virus is internalized into human cells by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor through its spike protein (S-glycoprotein). As S-glycoprotein is required for the attachment and entry into the human target cells, it is the primary mediator of SARS-CoV-2 infectivity. Currently, this glycoprotein has received considerable attention as a key component for the development of antiviral vaccines or biologics against SARS-CoV-2. Moreover, since the ACE2 receptor constitutes the main entry route for the SARS-CoV-2 virus, its soluble form could be considered as a promising approach for the treatment of coronavirus disease 2019 infection (COVID-19). Both S-glycoprotein and ACE2 are highly glycosylated molecules containing 22 and 7 consensus N-glycosylation sites, respectively. The N-glycan structures attached to these specific sites are required for the folding, conformation, recycling, and biological activity of both glycoproteins. Thus far, recombinant S-glycoprotein and ACE2 have been produced primarily in mammalian cells, which is an expensive process. Therefore, benefiting from a cheaper cell-based biofactory would be a good value added to the development of cost-effective recombinant vaccines and biopharmaceuticals directed against COVID-19. To this end, efficient protein synthesis machinery and the ability to properly impose post-translational modifications make microalgae an eco-friendly platform for the production of pharmaceutical glycoproteins. Notably, several microalgae (e.g., Chlamydomonas reinhardtii, Dunaliella bardawil, and Chlorella species) are already approved by the U.S. Food and Drug Administration (FDA) as safe human food. Because microalgal cells contain a rigid cell wall that could act as a natural encapsulation to protect the recombinant proteins from the aggressive environment of the stomach, this feature could be used for the rapid production and edible targeted delivery of S-glycoprotein and soluble ACE2 for the treatment/inhibition of SARS-CoV-2. Herein, we have reviewed the pathogenesis mechanism of SARS-CoV-2 and then highlighted the potential of microalgae for the treatment/inhibition of COVID-19 infection.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and β-human chorionic gonadotropin (β-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a β-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of β-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

Abstract 1379: Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line

Abstract Background: Myeloproliferative neoplasms (MPNs) are a type of chronic blood cancers. A subset are driven by frameshift mutations in the calreticulin gene (CALR). The CALR protein is a lectin chaperone and primarily assists in glycoprotein folding in the endoplasmic reticulum. It is also part of the peptide loading complex, a set of proteins involved in antigen processing and peptide presentation by major histocompatibility complex class I (MHC-I) molecules. All MPN-associated frameshift mutations in CALR lead to a similar change in its C-terminal, altering its protein-protein interactions. Examples include impaired chaperone activity of mutant CALR that impacts protein homeostasis and compromised activity of the MHC-I peptide loading complex. We hypothesized that these impaired functions will alter the repertoire of peptides presented by MHC-I in mutant CALR expressing cells, which we characterized in a patient-derived, mutant-CALR-driven myeloid tumor cell line, Marimo. Methods: We carried out an immunopeptidome analysis on Marimo cell line derivates expressing either wildtype or mutant CALR to identify potential MPN-associated peptides presented by MHC-I, and validated their immunogenicity in human samples. MHC-I-peptide complexes were immunoprecipitated from these Marimo cell line derivates, followed by peptide elution and analysis by mass spectrometry. A subset of peptides that were found in the mass spectrometry analysis of mutant CALR cells but not wildtype CALR cells were tested for their immunogenicity by assaying the in vitro T cell responses they evoked. Naïve T cells from healthy donors, carrying at least one MHC-I allele that matched the Marimo cells, were primed and expanded with the selected peptides and peptide-specific T cell responses were measured by intracellular staining detecting effector cytokines IFN-γ and TNF-α. Results: 1. The expression of mutant CALR disrupts the function of the peptide loading complex, leading to reduced recruitment of MHC-I and reducing its cell surface expression by almost 40% compared to cells expressing wildtype CALR. 2. Peptide repertoire of MHC-I is significantly altered by the expression of mutant CALR. Immunopeptidomics revealed a qualitative and quantitative difference in peptides presented by cells expressing wildtype CALR vs mutant CALR 3. The immunogenicity of a curated list of 24 peptides was tested in healthy donors, and at least 6 peptides activated T cell responses. Similar testing in MPN patients is planned. Conclusions: This is the first study to investigate the impact of mutant CALR on the repertoire of peptides presented by MHC-I in myeloid cells. We discovered tumor-associated, immunogenic peptides that may serve as potential targets of immunotherapy allowing the specific elimination of mutant CALR-expressing cells, which has been a challenge in the treatment of MPNs. Citation Format: Najla Arshad, Nathalie Vigneron, Cansu Cimen Bozkus, Vincent Stroobant, Stefan Naulaerts, Nina Bhardwaj, Benoît Van den Eynde, Peter Cresswell. Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1379.

Oligomannose-Type Glycan Processing in the Endoplasmic Reticulum and Its Importance in Misfolding Diseases.

Simple SummaryGlycans play many roles in biological processes. For instance, they mediate cell–cell interaction, viral infection, and protein folding of glycoproteins. Glycoprotein folding in the endoplasmic reticulum (ER) is closely related to the onset of diseases such as misfolding diseases caused by accumulation of misfolded proteins in the ER. In this review, we focused on oligomannose-type glycan processing in the ER, which has central roles in glycoprotein folding in the ER, and we summarise relationship between oligomannose-type glycan processing and misfolding diseases arising from the disruption of ER homeostasis.Glycoprotein folding plays a critical role in sorting glycoprotein secretion and degradation in the endoplasmic reticulum (ER). Furthermore, relationships between glycoprotein folding and several diseases, such as type 2 diabetes and various neurodegenerative disorders, are indicated. Patients’ cells with type 2 diabetes, and various neurodegenerative disorders induce ER stress, against which the cells utilize the unfolded protein response for protection. However, in some cases, chronic and/or massive ER stress causes critical damage to cells, leading to the onset of ER stress-related diseases, which are categorized into misfolding diseases. Accumulation of misfolded proteins may be a cause of ER stress, in this respect, perturbation of oligomannose-type glycan processing in the ER may occur. A great number of studies indicate the relationships between ER stress and misfolding diseases, while little evidence has been reported on the connection between oligomannose-type glycan processing and misfolding diseases. In this review, we summarize alteration of oligomannose-type glycan processing in several ER stress-related diseases, especially misfolding diseases and show the possibility of these alteration of oligomannose-type glycan processing as indicators of diseases.

A Quinolin-8-Ol Sub-Millimolar Inhibitor of UGGT, the ER Glycoprotein Folding Quality Control Checkpoint

A Quinolin-8-Ol Sub-Millimolar Inhibitor of UGGT, the ER Glycoprotein Folding Quality Control Checkpoint

Arabidopsis PDI11 interacts with lectin molecular chaperons calreticulin 1 and 2 through its D domain

The endoplasmic reticulum (ER) is equipped with protein disulfide isomerases (PDIs), molecular chaperons, and other folding enzymes to ensure that newly synthesized proteins in the ER are properly folded. Molecular chaperons and PDIs can form complex to promote protein folding in the ER of mammalian cells. In plants, many PDIs associate with each other and function cooperatively in oxidative protein folding. As a plant unique protein disulfide isomerase, Arabidopsis thaliana PDI11 (AtPDI11) demonstrates oxidative protein folding activities and works synergistically with AtPDI2/5. However, whether AtPDI11 associates with molecular chaperons or AtPDIs in catalyzing disulfide formation remained unknown. Here, we find that AtPDI11 interacts with ER resident lectin chaperones calreticulin 1 (CRT1) and CRT2. Furthermore, the D domain, but not the a or a' domain of AtPDI11 provides the biding sites for its interaction with CRT1/2. Moreover, the P domain of CRT1 is responsible for its interaction with AtPDI11. Our work implies that Arabidopsis CRT1/2 may specifically recruit AtPDI11 to assist the folding of glycoproteins in the ER.

N-Substituted Valiolamine Derivatives as Potent Inhibitors of Endoplasmic Reticulum α-Glucosidases I and II with Antiviral Activity.

Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.

N-acetylglucosaminyltransferase II Is Involved in Plant Growth and Development Under Stress Conditions.

Alpha-1,6-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase [EC 2.4.1.143, N-acetylglucosaminyltransferase II (GnTII)] catalyzes the transfer of N-acetylglucosamine (GlcNAc) residue from the nucleotide sugar donor UDP-GlcNAc to the α1,6-mannose residue of the di-antennary N-glycan acceptor GlcNAc(Xyl)Man3(Fuc)GlcNAc2 in the Golgi apparatus. Although the formation of the GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 N-glycan is known to be associated with GnTII activity in Arabidopsis thaliana, its physiological significance is still not fully understood in plants. To address the physiological importance of the GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 N-glycan, we examined the phenotypic effects of loss-of-function mutations in GnTII in the presence and absence of stress, and responsiveness to phytohormones. Prolonged stress induced by tunicamycin (TM) or sodium chloride (NaCl) treatment increased GnTII expression in wild-type Arabidopsis (ecotype Col-0) but caused severe developmental damage in GnTII loss-of-function mutants (gnt2-1 and gnt2-2). The absence of the 6-arm GlcNAc residue in the N-glycans in gnt2-1 facilitated the TM-induced unfolded protein response, accelerated dark-induced leaf senescence, and reduced cytokinin signaling, as well as susceptibility to cytokinin-induced root growth inhibition. Furthermore, gnt2-1 and gnt2-2 seedlings exhibited enhanced N-1-naphthylphthalamic acid-induced inhibition of tropic growth and development. Thus, GnTII’s promotion of the 6-arm GlcNAc addition to N-glycans is important for plant growth and development under stress conditions, possibly via affecting glycoprotein folding and/or distribution.