Induction of oxidative- and endoplasmic-reticulum-stress dependent apoptosis in pancreatic cancer cell lines by DDOST knockdown.

The dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit (DDOST) is a key component of the oligosaccharyltransferase complex catalyzing N-linked glycosylation in the endoplasmic reticulum lumen. DDOST is associated with several cancers and congenital disorders of glycosylation. However, its role in pancreatic cancer remains elusive, despite its enriched pancreatic expression. Using quantitative mass spectrometry, we identify 30 differentially expressed proteins and phosphopeptides (DEPs) after DDOST knockdown in the pancreatic ductal adenocarcinoma (PDAC) cell line PA-TU-8988T. We evaluated DDOST / DEP protein-protein interaction networks using STRING database, correlation of mRNA levels in pancreatic cancer TCGA data, and biological processes annotated to DEPs in Gene Ontology database. The inferred DDOST regulated phenotypes were experimentally verified in two PDAC cell lines, PA-TU-8988T and BXPC-3. We found decreased proliferation and cell viability after DDOST knockdown, whereas ER-stress, ROS-formation and apoptosis were increased. In conclusion, our results support an oncogenic role of DDOST in PDAC by intercepting cell stress events and thereby reducing apoptosis. As such, DDOST might be a potential biomarker and therapeutic target for PDAC.

STT3A-mediated viral N-glycosylation underlies the tumor selectivity of oncolytic virus M1.

Oncolytic viruses are emerging as promising anticancer agents. Although the essential biological function of N-glycosylation on viruses are widely accepted, roles of N-glycan and glycan-processing enzyme in oncolytic viral therapy are remain elusive. Here, via cryo-EM analysis, we identified three distinct N-glycans on the envelope of oncolytic virus M1 (OVM) as being necessary for efficient receptor binding. E1-N141-glycan has immediate impact on the binding of MXRA8 receptor, E2-N200-glycan mediates the maturation of E2 from its precursor PE2 which is unable to bind with MXRA8, and E2-N262-glycan slightly promotes receptor binding. The necessity of OVM N-glycans in receptor binding make them indispensable for oncolysis in vitro and in vivo. Further investigations identified STT3A, a key catalytic subunit of oligosaccharyltransferase (OST), as the determinant of OVM N-glycosylation, and STT3A expression in tumor cells is positively correlated with OVM-induced oncolysis. Increased STT3A expression was observed in various solid tumors, pointing to a broad-spectrum anticancer potential of OVM. Collectively, our research supports the importance of STT3A-mediated N-glycosylation in receptor binding and oncolysis of OVM, thus providing a novel predictive biomarker for OVM.

YTHDF1 Promotes Bladder Cancer Cell Proliferation via the METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR Axis.

N6-methyladenosine (m6A) is the most common mRNA modification and it plays a critical role in tumor progression, prognoses and therapeutic response. In recent years, more and more studies have shown that m6A modifications play an important role in bladder carcinogenesis and development. However, the regulatory mechanisms of m6A modifications are complex. Whether the m6A reading protein YTHDF1 is involved in the development of bladder cancer remains to be elucidated. The aims of this study were to determine the association between METTL3/YTHDF1 and bladder cancer cell proliferation and cisplatin resistance to explore the downstream target genes of METTL3/YTHDF1 and to explore the therapeutic implications for bladder cancer patients. The results showed that the reduced expression of METTL3/YTHDF1 could lead to decreased bladder cancer cell proliferation and cisplatin sensitivity. Meanwhile, overexpression of the downstream target gene, RPN2, could rescue the effect of reduced METTL3/YTHDF1 expression on bladder cancer cells. In conclusion, this study proposes a novel METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR regulatory axis that affects bladder cancer cell proliferation and cisplatin sensitivity.

RPN2 in cancer: An overview.

Oncogenes together with tumor suppresser genes are confirmed to regulate tumor phenotype in human cancers. RPN2, widely verified as an oncogene, encodes a protein that is part of an N-oligosaccharyl transferase, and is observed to be aberrantly expressed in human malignancies. Accumulating evidence unveils the vital functions of RPN2, contributing to tumorigenicity, metastasis, progression, and multi-drug resistance. Furthermore, previous studies partly indicated that RPN2 was involved in tumor progression via contributing to N-glycosylation and regulating multiple signaling pathways. In addition, RPN2 was also confirmed as a downstream target involved in tumor progression. Moreover, with demonstrated prognosis value and therapeutic target, RPN2 was also determined as a promising biomarker for forecasting patients' prognostic and therapy efficacy. In the present review, we aimed to summarize the present studies of RPN2 in cancer, and enhance the understanding of RPN2's extensive functions and clinical significances.

circTOP2A functions as a ceRNA to promote glioma progression by upregulating RPN2.

Competing endogenous RNA (ceRNA)-mediated signaling pathway dysregulation provides great insight into comprehensively understanding the molecular mechanism and combined targeted therapy for glioblastoma. circRNA is characterized by high stability, tissue/developmental stage-specific expression and abundance in brain and plays significant roles in the initiation and progression of cancer. Our previous published data have demonstrated that RPN2 was significantly upregulated in glioma and promoted tumor progression via the activation of the Wnt/ß-catenin pathway. Furthermore, we proved that miR-422a regulated the Wnt/ß-catenin signaling pathway by directly targeting RPN2. In this study, based on the glioblastoma microarray profiles, we identified the upstream circTOP2A, which completely bound to miR-422a and was co-expressed with the RPN2. circTOP2A was significantly overexpressed in glioma and conferred a poor prognosis. circTOP2A could regulate RPN2 expression by sponging miR-422a, verified by western blot, dual-luciferase reporter gene assay, and RNA pull-down assay. Functional assays including CCK8, transwell and FITC-annexin V were performed to explore the RPN2-mediated role of the circTOP2A effect on the glioma malignant phenotype. Additionally, TOP/FOP and immunofluorescence analysis were used to confirm that sh-circTOP2A could suppress the Wnt/ß-catenin pathway partly through RPN2. Finally, a tumor xenograft model was applied to validate the biological function of circTOP2A in vivo. Taken together, our findings reveal the critical role of circTOP2A in promoting glioma proliferation and invasion via a ceRNA mechanism and provide an exploitable biomarker and therapeutic target for glioma patients.

Identification and validation of a signature based on macrophage cell marker genes to predict recurrent miscarriage by integrated analysis of single-cell and bulk RNA-sequencing.

Recurrent miscarriage (RM) is a chronic, heterogeneous autoimmune disease that has serious social and personal consequences. No valid and reliable diagnostic markers or therapeutic targets for RM have been identified. Macrophages impact the innate immune system and can be used as diagnostic and prognostic markers for many diseases. We first collected 16 decidua and villi tissue samples from 5 normal patients and 3 RM patients for single-cell RNA sequencing data analysis and identified 1293 macrophage marker genes. We then screened a recurrent miscarriage cohort (GSE165004) for 186 macrophage-associated marker genes that were significantly differentially expressed between RM patients and the normal pregnancy endometrial tissues, and performed a functional enrichment analysis of differentially expressed genes. We then identified seven core genes (ACTR2, CD2AP, MBNL2, NCSTN, PUM1, RPN2, and TBC1D12) from the above differentially expressed gene group that are closely related to RM using the LASSO, Random Forest and SVM-RFE algorithms. We also used GSE26787 and our own collection of clinical specimens to further evaluate the diagnostic value of the target genes. A nomogram was constructed of the expression levels of these seven target genes to predict RM, and the ROC and calibration curves showed that our nomogram had a high diagnostic value for RM. These results suggest that ACTR2 and NCSTN may be potential targets for preventative RM treatments.

The N6-methyladenosine-mediated lncRNA WEE2-AS1 promotes glioblastoma progression by stabilizing RPN2.

Background: Glioblastoma (GBM) is the most common primary brain malignancy and has high aggressiveness and a poor prognosis. N6-methyladenosine (m6A) represents the most prevalent methylation modification of lncRNAs and has been shown to play important roles in the pathophysiological processes of tumors. However, the distribution and function of m6A modifications in lncRNAs in GBM tissues have not been fully revealed. Methods: The global depiction of m6A-modified lncRNA expression patterns in GBM tumor tissues was screened via m6A high-throughput sequencing. Gain- and loss-of-function assays were performed to investigate the role of WEE2-AS1 in GBM. Mass spectrometry and RNA-pulldown, RNA immunoprecipitation (RIP), luciferase reporter and coimmunoprecipitation assays were performed to explore the mechanism of m6A-mediated upregulation of WEE2-AS1 expression and the downstream mechanism promoting the malignant progression of GBM. Results: Herein, we report the differential expression profile of m6A-modified lncRNAs in human GBM tissues for the first time. WEE2-AS1 was identified as a novel m6A-modified lncRNA that promotes GBM progression and was post-transcriptionally stabilized by IGF2BP3, an m6A reader. Moreover, we confirmed that WEE2-AS1 promoted RPN2 protein stabilization by preventing CUL2-mediated RPN2 K322 ubiquitination, thereby contributing to GBM malignant progression by activating the PI3K-Akt signaling pathway. In translational medicine, we found that blocking WEE2-AS1 expression improved the therapeutic sensitivity of dasatinib, a central nervous system penetrant that is FDA-approved in GBM. Conclusions: Overall, this work highlights that WEE2-AS1 may serve as a potential prognostic biomarker and therapeutic target in GBM, the knockdown of which significantly improves the efficacy of dasatinib, providing a promising strategy for improving targeted combination therapy for GBM patients.

Purification of Aspergillus tamarii mycelial fructosyltransferase (m-FTase), optimized FOS production, and evaluation of its anticancer potential.

In the present study, generation of prebiotic fructooligosaccharides (FOS) using Aspergillus tamarii FTase was optimized by applying response surface methodology. Optimal FOS (251 g L-1 ) was generated at 28.4°C, pH 7.0 and 50% (w/v) sucrose leading to 1.97-fold yield enhancement. The m-FTase was purified using ultrafiltration followed by HiTrap Q HP anion exchange chromatography resulting in 2.15-fold purified FTase with 12.76 U mg-1 specific activity. Purified FTase (75 kDa) had Km and Vmax values of 1049.717 mM and 2.094 µmol min-1  mg-1 , respectively. FOS incorporation led to upregulation of caspase 3, caspase 9, and Bax genes suggesting mitochondrial apoptosis activation in cancer cells. The study describes characteristics of purified FTase from A. tamarii, production optimization of FOS and unravels the role of FOS in anticancer activity against HT-29 cells. PRACTICAL APPLICATION: This study provides detailed insights of kinetic and thermodynamic characteristics of purified FTase, a prebiotic FOS-generating enzyme. Moreover, the role of the apoptotic genes involved in anticancer activity, and the prebiotic potential of FOS is also investigated. These findings are important in the context of FOS applications, and the optimized production strategies make it useful for industrial application.

The Oligosaccharyltransferase AglB Supports Surface-Associated Growth and Iron Oxidation in Methanococcus maripaludis.

Most microbial organisms grow as surface-attached communities known as biofilms. However, the mechanisms whereby methanogenic archaea grow attached to surfaces have remained understudied. Here, we show that the oligosaccharyltransferase AglB is essential for growth of Methanococcus maripaludis strain JJ on glass or metal surfaces. AglB glycosylates several cellular structures, such as pili, archaella, and the cell surface layer (S-layer). We show that the S-layer of strain JJ, but not strain S2, is a glycoprotein, that only strain JJ was capable of growth on surfaces, and that deletion of aglB blocked S-layer glycosylation and abolished surface-associated growth. A strain JJ mutant lacking structural components of the type IV-like pilus did not have a growth defect under any conditions tested, while a mutant lacking the preflagellin peptidase (ΔflaK) was defective for surface growth only when formate was provided as the sole electron donor. Finally, for strains that are capable of Fe0 oxidation, we show that deletion of aglB decreases the rate of anaerobic Fe0 oxidation, presumably due to decreased association of biomass with the Fe0 surface. Together, these data provide an initial characterization of surface-associated growth in a member of the methanogenic archaea. IMPORTANCE Methanogenic archaea are responsible for producing the majority of methane on Earth and catalyze the terminal reactions in the degradation of organic matter in anoxic environments. Methanogens often grow as biofilms associated with surfaces or partner organisms; however, the molecular details of surface-associated growth remain uncharacterized. We have found evidence that glycosylation of the cell surface layer is essential for growth of M. maripaludis on surfaces and can enhance rates of anaerobic iron corrosion. These results provide insight into the physiology of surface-associated methanogenic organisms and highlight the importance of surface association for anaerobic iron corrosion.

Computational Design of Oligosaccharide-Producing Levansucrase from Bacillus licheniformis RN-01 to Increase Its Stability at High Temperature.

Levan-type fructooligosaccharides (LFOs) and levan can potentially be used as ingredients in prebiotics, skincare products, and antitumor agents. The Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide-producing levansucrase, OPL) was reported to productively synthesize LFOs; however, OPL's thermostability is low at high temperatures. To enhance OPL structural stability, this study employed molecular dynamics (AMBER) to identify a highly flexible region, as measured by its average root-mean-square fluctuation (RMSF) value, on the OPL surface and computational protein design (Rosetta) to rigidify and increase favorable interactions to increase its structural stability. AMBER identified region nine (residues 277-317) as a highly flexible region that was selected for design because it has the highest number of residues and the second-highest average RMSF, and it is farthest from the active site. Rosetta designed 14 mutants with the best ΔΔG value in each position, where three mutants have better ΔG than OPL. To determine whether their flexibilities and stabilities are lower than those of OPL, all 14 designed mutants were simulated at high temperature (500 K), and we found that K296E, G309S, and A310W mutants were predicted to be more stable and could retain their native structures better than OPL. Our results suggest that enhanced structural stabilities of these mutants are caused by increased hydrogen bond strengths of the designed residues and their neighboring residues. This study designed K296E, G309S, and A310W mutants of OPL with high potential for stability improvement, and they could potentially be used for the effective production of LFOs.

Optimization strategy of Bacillus subtilis MT453867 levansucrase and evaluation of levan role in pancreatic cancer treatment.

Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects.

MALAT-1: LncRNA ruling miR-182/PIG-C/mesothelin triad in triple negative breast cancer.

Breast cancer (BC) remains a major health problem, despite the remarkable advances in cancer research setting. BC is the most common cancer affecting women worldwide. In the context of triple negative breast cancer (TNBC) treatment, major obstacles include late diagnoses and detrimental side effects of chemotherapy and radiotherapy. Research effort was rewarded with the discovery of mesothelin (MSLN), an oncogenic Glycosyl-Phosphatidyl-Inositol (GPI) anchored protein, over-expressed in TNBC. GPI pathway is a post-translational modification that attaches proteins to cellular membrane. MSLN targeted therapy succeeded in early clinical trials, nevertheless, to date, the epigenetic regulation of MSLN and GPI pathway by non-coding RNAs (nc-RNAs) in BC remains an untouched area. Accordingly, our aim is to investigate-for the first time- the impact of simultaneous targeting of MSLN and its associated GPI pathway member, PIG-C, by non-coding-RNAs. Expression profiling of PIG-C, MSLN in BC was performed. Using bioinformatics tools, MALAT-1 and miR-182 were found to target MSLN and PIG-C. MDA-MB-231 cells were transfected with synthetic nc-RNAs. Expression profiling of MSLN, miR-182 and MALAT-1 showed a dramatic over-expression in BC samples. MiR-182 ectopic expression and MALAT-1 silencing increased MSLN and PIG-C transcript levels. However, miR-182 inhibition and miR-182/si-MALAT-1 co-transfection lowered MSLN and PIG-C levels. Finally, si-PIG-C decreased MSLN and PIG-C levels. To conclude, our investigation unravels a new axis in TNBC, where miR-182 can manipulate MSLN and PIG-C. Meanwhile, MALAT-1 is the culprit lncRNA in this novel axis, possibly a sponge for miR-182. Altogether, this sheds light on new targets for BC immune-therapy.

Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin-avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle.

The AT1 receptor autoantibody causes hypoglycemia in fetal rats via promoting the STT3A-GLUT1-glucose uptake axis in liver.

Blood glucose is of great importance to development and metabolic homeostasis in fetuses. Stimulation of harmful factors during gestation induces pathoglycemia. Angiotensin II type 1 receptor autoantibody (AT1-AA), a newly discovered gestational harmful factor, has been shown to induce intrauterine growth restriction in fetuses and glucose disorders in adults. However, whether and how AT1-AA influences the blood glucose level of fetuses during gestation is not yet clear. The purpose of the current study was to observe the fetal blood glucose level of AT1-AA-positive pregnant rats during late pregnancy and to determine the roles that hepatic glucose transporters play in this process. We established AT1-AA-positive pregnant rats by injecting AT1-AA into the caudal veins of rats in the 2nd trimester of gestation. Although the fetal blood glucose level in the 3rd trimester of gestation decreased, hepatic glucose uptake increased detected. Through separating membrane and cytosolic proteins, we demonstrated that both the expression and membrane transport ratio of glucose transporter 1 (GLUT1), which is responsible for glucose transport in fetal hepatocytes, were upregulated, accompanied by increased expression of N-glycosyltransferase STT3A, which contributes to the N-glycosylation of GLUT1. In vitro, we identified that AT1-AA increased glucose uptake, the expression and membrane transport ratio of GLUT1 and the expression of STT3A in HepG2 cell lines via separating membrane and cytosolic proteins and immunofluorescence, resulting in the decreased glucose content in the medium. The GLUT1 inhibitor WZB117 reversed the decreases in glucose content in the medium, the increases in glucose uptake, the increases in the expression and membrane transport ratio of GLUT1 caused by AT1-AA. The N-glycosyltransferase inhibitor NGI as well as si-STT3A reversed the AT1-AA-induced upregulation of the STT3A-GLUT1-glucose uptake effect. This study demonstrates that AT1-AA lowers the blood glucose level of fetuses via the STT3A-GLUT1-glucose uptake axis in liver.

Inflammatory conditions promote a switch of oligosaccharyltransferase (OST) catalytic subunit isoform expression.

Oligosaccharyltransferase (OST) complex catalyzes the N-glycosylation of nascent polypeptides in the endoplasmic reticulum. Glycoproteins are critical for normal cell-cell interactions, especially during an immune response. Abnormal glycosylation is an insignia of several inflammatory diseases. However, the mechanisms that regulate the differential N-glycosylation are not fully understood. The OST complex can be assembled with one out of two catalytic subunits, STT3A or STT3B, which have different enzymatic properties. In this work, we investigated the expression of STT3A and STT3B in several mouse models such as a crossbreeding of normal and abortion-prone mice and an intestinal inflammation model. These animals were either exposed or not to acoustic stress (acute or chronic). The expression of the isoforms was analysed by immunohistochemistry and protein immunoblot. Finally, we investigated the gene regulatory elements employing public databases. Results demonstrated that inflammation alters the balance between the expression of both isoforms in the affected tissues. In homoeostatic conditions, STT3A expression predominates over STT3B, especially in epithelial cells. This relation is reversed as a consequence of inflammation. An increase in STT3B activity was associated to the generation of mannose-rich N-glycans. Accordingly, this type of N-glycans were found to decorate diverse inflamed tissues. The STT3A and STT3B genes are differentially regulated, which could account for the differences in the expression levels observed here. Our results support the idea that these isoforms could play different roles in cellular physiology. This study opens the possibility of studying the STT3A/STT3B expression ratio as a biomarker in acute inflammation or chronic diseases.

Overexpression of RPN2 suppresses radiosensitivity of glioma cells by activating STAT3 signal transduction.

BACKGROUND: Radiation therapy is the primary method of treatment for glioblastoma (GBM). Therefore, the suppression of radioresistance in GBM cells is of enormous significance. Ribophorin II (RPN2), a protein component of an N-oligosaccharyl transferase complex, has been associated with chemotherapy drug resistance in multiple cancers, including GBM. However, it remains unclear whether this also plays a role in radiation therapy resistance in GBM. METHODS: We conducted a bioinformatic analysis of RPN2 expression using the UCSC Cancer Genomics Browser and GEPIA database and performed an immunohistochemical assessment of RPN2 expression in biopsy specimens from 34 GBM patients who had received radiation-based therapy. We also studied the expression and function of RPN2 in radiation-resistant GBM cells. RESULTS: We found that RPN2 expression was upregulated in GBM tumors and correlated with poor survival. The expression of RPN2 was also higher in GBM patients with tumor recurrence, who were classified to be resistant to radiation therapy. In the radiation-resistant GBM cells, the expression of RPN2 was also higher than in the parental cells. Depletion of RPN2 in resistant cells can sensitize these cells to radiation-induced apoptosis, and overexpression of RPN2 had the reverse effect. Myeloid cell leukemia 1 (MCL1) was found to be the downstream target of RPN2, and contributed to radiation resistance in GBM cells. Furthermore, STAT3 was found to be the regulator of MCL1, which can be activated by RPN2 dysregulation. CONCLUSION: Our study has revealed a novel function of RPN2 in radiation-resistant GBM, and has shown that MCL1 depletion or suppression could be a promising method of therapy to overcome the resistance promoted by RPN2 dysregulation.

N-Glycosylation Regulates Chitinase 3-like-1 and IL-13 Ligand Binding to IL-13 Receptor α2.

Chitinase 3-like-1 (Chi3l1) and IL-13 are both ligands of IL-13 receptor α2 (IL-13Rα2). The binding of the former activates mitogen-activated protein kinase, AKT, and Wnt/ß-catenin signaling, and plays important roles in innate and adaptive immunity, cellular apoptosis, oxidative injury, allergic inflammation, tumor metastasis and wound healing, fibrosis, and repair in the lung. In contrast, the latter binding is largely a decoy event that diminishes the effects of IL-13. Here, we demonstrate that IL-13Rα2 N-glycosylation is a critical determinant of which ligand binds. Structure-function evaluations demonstrated that Chi3l1-IL-13Rα2 binding was increased when sites of N-glycosylation are mutated, and studies with tunicamycin and Peptide:N-glycosidase F (PNGase F) demonstrated that Chi3l1-IL-13Rα2 binding and signaling were increased when N-glycosylation was diminished. In contrast, structure-function experiments demonstrated that IL-13 binding to IL-13Rα2 was dependent on each of the four sites of N-glycosylation in IL-13Rα2, and experiments with tunicamycin and PNGase F demonstrated that IL-13-IL-13Rα2 binding was decreased when IL-13Rα2 N-glycosylation was diminished. Studies with primary lung epithelial cells also demonstrated that Chi3l1 inhibited, whereas IL-13 stimulated, N-glycosylation as evidenced by the ability of Chi3l1 to inhibit and IL-13 to stimulate the subunits of the oligosaccharide complex A and B (STT3A and STT3B). These studies demonstrate that N-glycosylation is a critical determinant of Chi3l1 and IL-13 binding to IL-13Rα2, and highlight the ability of Chi3l1 and IL-13 to alter key elements of the N-glycosylation apparatus in a manner that would augment their respective binding.

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase.

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell-cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

Overexpression of RPN2 promotes osteogenic differentiation of hBMSCs through the JAK/STAT3 pathway.

Osteoporosis is characterized by decreased bone mass and degenerating bone structure, which cause severe bone fragility and increase the risk for fractures. Human bone mesenchymal stem cells (hBMSCs) differentiate into osteoblasts through osteogenesis, and disturbances in the balance between bone generation and degeneration underlie the pathogenesis of senile osteoporosis. The highly conserved glycoprotein Ribophorin II (RPN2) is involved in multiple biological reactions, but the role of RPN2 in the osteogenic differentiation of hBMSCs and their molecular etiology is incompletely understood. Here, we show that RPN2 expression is up-regulated in hBMSCs during osteogenic differentiation. In vitro assays revealed that silencing of RPN2 inhibited hBMSC differentiation into osteoblasts. Moreover, RPN2 overexpression enhanced the expression of linked genes and resulted in high alkaline phosphatase activity. Our results suggest that RPN2 targets Janus kinase 1 (JAK1), and RPN2 overexpression was observed to induce JAK1 ubiquitination. Depletion of JAK1 facilitated osteogenic differentiation of RPN2-silenced hBMSCs. Moreover, western blot analysis revealed that RPN2 silencing suppressed the stimulation and nuclear translocation of the downstream signal transducer and activator of transcription 3 sensor; this could be reversed via RPN2 overexpression. This research sheds light on an innovative molecular mechanism that is associated with hBMSC differentiation into osteoblasts and may facilitate bone anabolism through RPN2.

Oligosaccharyltransferase: A Gatekeeper of Health and Tumor Progression.

Oligosaccharyltransferase (OST) is a multi-span membrane protein complex that catalyzes the addition of glycans to selected Asn residues within nascent polypeptides in the lumen of the endoplasmic reticulum. This process, termed N-glycosylation, is a fundamental post-translational protein modification that is involved in the quality control, trafficking of proteins, signal transduction, and cell-to-cell communication. Given these crucial roles, N-glycosylation is essential for homeostasis at the systemic and cellular levels, and a deficiency in genes that encode for OST subunits often results in the development of complex genetic disorders. A growing body of evidence has also demonstrated that the expression of OST subunits is cell context-dependent and is frequently altered in malignant cells, thus contributing to tumor cell survival and proliferation. Importantly, a recently developed inhibitor of OST has revealed this enzyme as a potential target for the treatment of incurable drug-resistant tumors. This review summarizes our current knowledge regarding the functions of OST in the light of health and tumor progression, and discusses perspectives on the clinical relevance of inhibiting OST as a tumor treatment.