A review of fructosyl-transferases from catalytic characteristics and structural features to reaction mechanisms and product specificity.

Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and ß-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed ß-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and ß-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS.

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.

Structural and mechanistic studies of the N-glycosylation machinery: from lipid-linked oligosaccharide biosynthesis to glycan transfer.

N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a lipid-linked oligosaccharide (LLO), and (ii) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided an insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.

Characterization and alteration of product specificity of Beijerinckia indica subsp. indica ß-fructosyltransferase.

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a ß-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180 g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation.

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.

A genome-wide CRISPR functional survey of the human phagocytosis molecular machinery.

Phagocytosis, the process by which cells engulf large particles, plays a vital role in driving tissue clearance and host defense. Its dysregulation is connected to autoimmunity, toxic accumulation of proteins, and increased risks for infections. Despite its importance, we lack full understanding of all molecular components involved in the process. To create a functional map in human cells, we performed a genome-wide CRISPRko FACS screen that identified 716 genes. Mapping those hits to a comprehensive protein-protein interaction network annotated for functional cellular processes allowed retrieval of protein complexes identified multiple times and detection of missing phagocytosis regulators. In addition to known components, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, we identified and validated new phagocytosis-relevant functions, including the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) and the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our phagocytosis network comprises elements of cargo uptake, shuffling, and biotransformation through the cell, providing a resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. Our approach of integrating protein-protein interaction offers a broadly applicable way to functionally interpret genome-wide screens.

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.

Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

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.

Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV.

N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

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.

Proteome and Glycoproteome Analyses Reveal the Protein N-Linked Glycosylation Specificity of STT3A and STT3B.

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensis HL12 on Short-Chain Levan-Type Fructooligosaccharides Production.

Levan-type fructooligosaccharides (L-FOSs) are a prominent class of non-digestible oligosaccharides with potential as nutritional prebiotics. Endo-levanase, which randomly hydrolyzes ß-(2,6)-linkages in fructans, is a promising enzyme for short-chain FOS production. In this work, a recombinant levanase (LevBk) from Bacillus koreensis strain HL12 was characterized. Soluble LevBk protein was produced in Escherichia coli BL21(DE3) system at 40 mg/L of culture medium. Based on sequence and structural analysis, LevBk was classified as a member of endo-levanase in GH32 family containing N-terminal substrate binding pocket and C-terminal ß-sandwich domains. LevBk optimally worked at 45 °C, pH 6.0 with the specific activity of 2.43 U/mg. Based on enzymatic hydrolysis, short-chain L-FOSs with degree of polymerization (DP) of 2-4 were produced from hydrolysis of timothy grass levan under optimal conditions for 9-24 h. With its ability to produce L-FOSs with specific chain lengths, LevBk could be attractively applied for converting of levan containing material to high value-added sweetener in the biorefinery industry.

Thermostability engineering of an inulin fructotransferase for the biosynthesis of difructose anhydride I.

The thermostability of enzymes is an essential factor that performs a vital role during practical applications. Inulin fructotransferases can efficiently convert inulin into bio-functional difructose anhydrides (DFAs). The present study aimed to improve the thermostability of a previously reported inulin fructotransferase, SpIFTase, and apply it to the biosynthesis of DFA I. In silico rational design was used to predict mutation sites, based on sequential and structural information. Two triple-site mutants, Q69L/Q234L/K310G and E201I/Q234L/K310G, were characterized and exhibited enhanced thermostability with approximately 5 °C higher in melting temperature (Tm), respectively, and a 45-fold longer half-life (t1/2) at 70 °C, compared to that of SpIFTase. Molecular dynamic simulations and elaborate structural analysis suggested that the combinations of hydrophobic interaction, electrostatic potential distribution, and decreased flexibility via stabilization of loops and α-helix improved the thermostability of SpIFTase. Additionally, the promising mutants exhibited great potential to the industrial production of DFA I.

Upregulation of ribosome complexes at the blood-brain barrier in Alzheimer's disease patients.

The cerebrovascular-specific molecular mechanism in Alzheimer's disease (AD) was investigated by employing comprehensive and accurate quantitative proteomics. Highly purified brain capillaries were isolated from cerebral gray and white matter of four AD and three control donors, and examined by SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25, and RPSA) were significantly upregulated in AD patients. This upregulation of ribosomal protein expression occurred only in brain capillaries and not in brain parenchyma. The protein expression of protein processing and N-glycosylation-related proteins in the endoplasmic reticulum (DDOST, STT3A, MOGS, GANAB, RPN1, RPN2, SEC61B, UGGT1, LMAN2, and SSR4) were also upregulated in AD brain capillaries and was correlated with the expression of ribosomal proteins. The findings reported herein indicate that the ribosome complex, the subsequent protein processing and N-glycosylation-related processes are significantly and specifically upregulated in the brain capillaries of AD patients.

Production of Human ABC Transporters and Oligosaccharyltransferase Complexes for Structural Studies.

Structural studies of membrane proteins require high-quality samples. The target proteins should not only be pure and homogeneous but should also be active and allow the capture of a functionally relevant state. Here we present optimized methods for the expression and purification of human ABC transporters and oligosaccharyltransferase (OST) complexes that can be used for high-resolution structure determination using single-particle cryo-electron microscopy (cryo-EM). The protocols are based on the generation of stable cell lines that enable tetracycline-inducible expression of the target proteins. For the multidrug exporter ABCB1, we describe a protocol for reconstitution into nanodiscs and evaluation of the ATPase activity in the presence of drugs. For human OST, we describe a strategy for the purification of OST-A and OST-B complexes, including techniques to evaluate their integrity and activity using in vitro glycosylation assays. These protocols can be adapted for the production of other human ABC transporters and multimeric membrane protein complexes.

Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose.

Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.

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.