Specific Glycosyltransferases Research Articles

Roles of Glyco-redox in Epithelial Mesenchymal Transition and Mesenchymal Epithelial Transition, Cancer, and Various Diseases.

Reduction-oxidation (redox) regulation is an important biological phenomenon that provides a balance between antioxidants and the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) under pathophysiological conditions. Structural and functional changes in glycans are also important as post-translational modifications of proteins. The integration of glycobiology and redox biology, called Glyco-redox has provided new insights into the mechanisms of epithelial-mesenchymal transition (EMT)/mesenchymal-epithelial transition (MET), cancer, and various diseases including Alzheimer's disease (AD), chronic obstructive lung disease (COPD), type 2 diabetes, interstitial pneumonitis, and ulcerative colitis (UC), . Glycans are biosynthesized by specific glycosyltransferases and each glycosyltransferase is either directly or indirectly regulated by oxidative stress and redox regulation. A typical example of Glyco-redox is the role of N-glycan referred to as core fucose in superoxide dismutase 3 (SOD3). This glycan was found to be involved in the growth inhibition of cancer cell lines. The significance of Glyco-redox in EMT/MET, cancer and various diseases was found in major N-glycan branching glycosyltransferases GnT-III, GnT-IV, GnT-V, VI, GnT-IX, Fut8, and ST6Gal1. Herein, we summarize previous reports on the target proteins and how this relates to oxidative stress. We also discuss the products of these processes and their significance to cancer and various diseases.

Glycosphingolipids in Osteoarthritis and Cartilage-Regeneration Therapy: Mechanisms and Therapeutic Prospects Based on a Narrative Review of the Literature.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell-surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolisms and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

Microbial Synthesis of Lacto-N-fucopentaose I with High Titer and Purity by Screening of Specific Glycosyltransferase and Elimination of Residual Lacto-N-triose II and Lacto-N-tetraose.

Lacto-N-fucopentaose I (LNFP I) has recently been approved as generally recognized as safe, demonstrating its great commercial potential in the food industry. Microbial synthesis through metabolic engineering strategies is an effective approach for large-scale production of LNFP I. Biosynthesis of LNFP I requires consideration of two key points: high titer with low byproduct 2'-fucosyllactose (2'-FL) generation and high purity with low lacto-N-triose II (LNTri II) and lacto-N-tetraose (LNT) residues. Herein, α1,2-fucosyltransferase from Thermoanaerobacterium sp. RBIITD was screened from 16 selected LNFP I-producing glycosyltransferase candidates, showing the highest in vivo LNFP I productivity. Chromosomal integration of wbgO enhanced the LNFP I production by improving the precursor conversion from LNTri II to LNT. The best engineered strain produced 4.42 and 35.1 g/L LNFP I in shake-flask and fed-batch cultivation, respectively. The residual LNTri II and LNT were eliminated by further cultivation with a recombinant strain coexpressing Bifidobacterium bifidum β-N-acetylhexosaminidase and lacto-N-biosidase. A strategy for LNFP I biosynthesis with high yield and purity was finally realized, providing support for its practical application in large-scale production.

ABO Blood System: Biosynthesis of Agglutinogenic Alkaline and Non-Agglutinogenic Acid Glycotopes of A and B Antigens at Different pHs of the Culture Medium.

The biosynthesis of agglutinogenic and adsorbing groups A and B glycotopes of the erythrocyte's membrane is mediated by the activity of specific glycosyltransferases. This study aimed to assess the nature of the biosynthesis of A and B antigenic glycotopes, depending on the pH of the medium during the cultivation of erythrocytes, and the antigenic (transferase) characteristics of the donor serum of the other group. Monoclonal antibodies (Mabs) were obtained from IGBRL under Program IV of the International Workshop on Monoclonal Antibodies and Red Blood Cell Antigens. Biosynthesis was performed using erythrocytes, fresh serum, medium 199, and an antibiotic solution. The agglutinogenic characteristics of 11 out of 33 samples changed by the end of the cultivation period due to the acquisition of additional agglutinogen corresponding to the donor serum. None of the samples lost their inherent agglutinogen due to its absence in the donor serum. Four of six samples of O(I) erythrocytes acquired the ability to be agglutinated by anti-A reagents, especially by the polyclonal anti-A, and the manifestation of agglutination depended on the reaction time. Two of the three samples with initial A(II) agglutinogenic specificity added to the donor serum with Bc'+ characteristic of the erythrocytes acquired this characteristic. However, none of the five A(II)Ac'+ samples cultured in the serum of Ac'-O(I)Ac'-Bc'+ and O(I)Ac'-Bc'- donors lost their inherent earlier Ac'+ characteristic. The investigation of the inhibitory ability of alkaline and acidic glycoconjugates isolated from membranes revealed that alkaline Alp-00 and Alp-1 glycotopes isolated from glycolipids had the highest inhibitory activity, and the degree of inhibition of polyclonal anti-A antibodies was even higher than that of monovalent BRIC-131. This study showed the possibility of the biosynthesis of specific non-agglutinogenic A and B glycotopes under the influence of a different group's serum as a source of the corresponding transferase.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using invitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

A sialyltransferases-related gene signature serves as a potential predictor of prognosis and therapeutic response for bladder cancer

BackgroundAberrant glycosylation, catalyzed by the specific glycosyltransferase, is one of the dominant features of cancers. Among the glycosyltransferase subfamilies, sialyltransferases (SiaTs) are an essential part which has close linkages with tumor-associated events, such as tumor growth, metastasis and angiogenesis. Considering the relationship between SiaTs and cancer, the current study attempted to establish an effective prognostic model with SiaTs-related genes (SRGs) to predict patients’ outcome and therapeutic responsiveness of bladder cancer.MethodsRNA-seq data, clinical information and genomic mutation data were downloaded (TCGA-BLCA and GSE13507 datasets). The comprehensive landscape of the 20 SiaTs was analyzed, and the differentially expressed SiaTs-related genes were screened with “DESeq2” R package. ConsensusClusterPlus was applied for clustering, following with survival analysis with Kaplan–Meier curve. The overall survival related SRGs were determined with univariate Cox proportional hazards regression analysis, and the least absolute shrinkage and selection operator (LASSO) regression analysis was performed to generate a SRGs-related prognostic model. The predictive value was estimated with Kaplan–Meier plot and the receiver operating characteristic (ROC) curve, which was further validated with the constructed nomogram and decision curve.ResultsIn bladder cancer tissues, 17 out of the 20 SiaTs were differentially expressed with CNV changes and somatic mutations. Two SiaTs_Clusters were determined based on the expression of the 20 SiaTs, and two gene_Clusters were identified based on the expression of differentially expressed genes between SiaTs_Clusters. The SRGs-related prognostic model was generated with 7 key genes (CD109, TEAD4, FN1, TM4SF1, CDCA7L, ATOH8 and GZMA), and the accuracy for outcome prediction was validated with ROC curve and a constructed nomogram. The SRGs-related prognostic signature could separate patients into high- and low-risk group, where the high-risk group showed poorer outcome, more abundant immune infiltration, and higher expression of immune checkpoint genes. In addition, the risk score derived from the SRGs-related prognostic model could be utilized as a predictor to evaluate the responsiveness of patients to the medical therapies.ConclusionsThe SRGs-related prognostic signature could potentially aid in the prediction of the survival outcome and therapy response for patients with bladder cancer, contributing to the development of personalized treatment and appropriate medical decisions.

MiR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis

BackgroundZoonotic microorganisms are increasingly impacting human health worldwide. Due to the development of the global population, humans and animals live in shared and progressively crowded ecosystems, which enhances the risk of zoonoses. Although Campylobacter species are among the most important bacterial zoonotic agents worldwide, the molecular mechanisms of many host and pathogen factors involved in colonisation and infection are poorly understood. Campylobacter jejuni colonises the crypts of the human colon and causes acute inflammatory processes. The mucus and associated proteins play a central host-protective role in this process. The aim of this study was to explore the regulation of specific glycosyltransferase genes relevant to differential mucin-type O-glycosylation that could influence host colonisation and infection by C. jejuni.ResultsSince microRNAs are known to be important regulators of the mammalian host cell response to bacterial infections, we focussed on the role of miR-125a-5p in C. jejuni infection. Combining in vitro and in vivo approaches, we show that miR-125a-5p regulates the expression of the sialyltransferase ST3GAL1 in an infection-dependent manner. The protein ST3GAL1 shows markedly increased intestinal levels in infected mice, with enhanced distribution in the mucosal epithelial layer in contrast to naïve mice.ConclusionFrom our previous studies and the data presented here, we conclude that miR-125a-5p and the previously reported miR-615-3p are involved in regulating the glycosylation patterns of relevant host cell response proteins during C. jejuni infection. The miRNA-dependent modulation of mucin-type O-glycosylation could be part of the mucosal immune response, but also a pathogen-driven modification that allows colonisation and infection of the mammalian host.

N-linked glycans: an underappreciated key determinant of T cell development, activation, and function.

N-linked glycosylation is a post-translational modification that results in the decoration of newly synthesized proteins with diverse types of oligosaccharides that originate from the amide group of the amino acid asparagine. The sequential and collective action of multiple glycosidases and glycosyltransferases are responsible for determining the overall size, composition, and location of N-linked glycans that become covalently linked to an asparagine during and after protein translation. A growing body of evidence supports the critical role of N-linked glycan synthesis in regulating many features of T cell biology, including thymocyte development and tolerance, as well as T cell activation and differentiation. Here, we provide an overview of how specific glycosidases and glycosyltransferases contribute to the generation of different types of N-linked glycans and how these post-translational modifications ultimately regulate multiple facets of T cell biology.

Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering.

Virtually all living cells are covered with glycans. Their structures are primarily controlled by the specificities of glycosyltransferases (GTs). GTs typically adopt one of the three folds, namely, GT-A, GT-B, and GT-C. However, what defines their specificities remain poorly understood. Here, we developed a genetic glycoengineering platform by reprogramming the capsular polysaccharide pathways in Streptococcus pneumoniae to interrogate GT specificity and manipulate glycan structures. Our findings suggest that the central cleft of GT-B enzymes is important for determining acceptor specificity. The constraint of the glycoengineering platform was partially alleviated when the specificity of the precursor transporter was reduced, indicating that the transporter contributes to the overall fidelity of glycan synthesis. We also modified the pneumococcal capsule to produce several medically important mammalian glycans, as well as demonstrated the importance of regiochemistry in a glycosidic linkage on binding lung epithelial cells. Our work provided mechanistic insights into GT specificity and an approach for investigating glycan functions.

An Overview of Sugar Nucleotide-Dependent Glycosyltransferases for Human Milk Oligosaccharide Synthesis.

Human milk oligosaccharides (HMOs) have received increasing attention because of their special effects on infant health and commercial value as the new generation of core components in infant formula. Currently, large-scale production of HMOs is generally based on microbial synthesis using metabolically engineered cell factories. Introduction of the specific glycosyltransferases is essential for the construction of HMO-producing engineered strains in which the HMO-producing glycosyltransferases are generally sugar nucleotide-dependent. Four types of glycosyltransferases have been used for typical glycosylation reactions to synthesize HMOs. Soluble expression, substrate specificity, and regioselectivity are common concerns of these glycosyltransferases in practical applications. Screening of specific glycosyltransferases is an important research topic to solve these problems. Molecular modification has also been performed to enhance the catalytic activity of various HMO-producing glycosyltransferases and to improve the substrate specificity and regioselectivity. In this article, various sugar nucleotide-dependent glycosyltransferases for HMO synthesis were overviewed, common concerns of these glycosyltransferases were described, and the future perspectives of glycosyltransferase-related studies were provided.

Bacterial extracellular polymeric substances: Biosynthesis and interaction with environmental pollutants

Extracellular polymeric substances (EPS) are highly hydrated matrices produced by bacteria, containing various polymers such as polysaccharides, proteins, lipids, and DNA. Extracellular polymer concentrations, ions, and functional groups provide physical stability to the EPS. Constituents of EPS form the three-dimensional architecture and help acquire nutrition for the bacteria. Structural and functional diversity of the extracellular polymer depends on the specific glycosyltransferases, polymerase and transporter proteins. These enzymes are encoded by specific genes present in operons such as crd, alg, wca, and gum reported in Agrobacterium, Pseudomonas, Enterobacteriaceae, and Xanthomonas. The operons regulate the biosynthesis of extracellular polymers such as curdlan, alginate, colonic acid, and xanthan, respectively. Various functional groups in the EPS, such as carbonyl, hydroxyl, phosphoryl, and amide, provide the sorption site for interaction with environmental pollutants. Hydrophobic interactions and coordinate bonds mainly dominate the binding of EPS with environmental pollutants. EPS binds, emulsifies, and solubilizes the organic compounds, enhancing the degradation process. EPS binds with heavy metals through complexation, surface adsorption, precipitation, and ion exchange mechanisms. The biodegradability efficiency and nontoxicity properties of EPS make it an excellent biopolymer for decontaminating environmental pollutants. This review summarizes an overview of the biosynthetic mechanisms and interaction of the bacterial extracellular polymer with environmental pollutants. Interaction mechanisms of pollutants with EPS and EPS-mediated bioremediation will help develop removal applications. Moreover, understanding the genes responsible for EPS production, and implementation of new genetic methodology can be helpful for the enhanced biosynthesis of EPS to control pollution by sequestrating more environmental pollutants.

N-Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling.

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.

Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disease that has been suffering from stagnant survival curves for decades. In the endeavor toward improved diagnosis and treatment, cellular glycosylation has emerged as an interesting focus area in AML. While mechanistic insights are still limited, aberrant glycosylation may affect intracellular signaling pathways of AML blasts, their interactions within the microenvironment, and even promote chemoresistance. Here, we performed a meta-omics study to portray the glycomic landscape of AML, thereby screening for potential subtypes and responsible glyco-regulatory networks.ResultsInitially, by integrating comprehensive N-, O-, and glycosphingolipid (GSL)-glycomics of AML cell lines with transcriptomics from public databases, we were able to pinpoint specific glycosyltransferases (GSTs) and upstream transcription factors (TFs) associated with glycan phenotypes. Intriguingly, subtypes M5 and M6, as classified by the French-American-British (FAB) system, emerged with distinct glycomic features such as high (sialyl) Lewisx/a ((s)Lex/a) and high sialylation, respectively. Exploration of transcriptomics datasets of primary AML cells further substantiated and expanded our findings from cell lines as we observed similar gene expression patterns and regulatory networks that were identified to be involved in shaping AML glycan signatures.ConclusionsTaken together, our data suggest transcriptionally imprinted glycomic signatures of AML, reflecting their differentiation status and FAB classification. This study expands our insights into the emerging field of AML glycosylation and paves the way for studies of FAB class-associated glycan repertoires of AML blasts and their functional implications.

Biosynthesis of Human Milk Oligosaccharides: Enzyme Cascade and Metabolic Engineering Approaches

Human milk oligosaccharides (HMOs) have unique beneficial effects for infants and are considered as the new gold standard for premium infant formula. They are a collection of unconjugated glycans, and more than 200 distinct structures have been identified. Generally, HMOs are enzymatically produced by elongation and/or modification from lactose via stepwise glycosylation. Each glycosylation requires a specific glycosyltransferase (GT) and the corresponding nucleotide sugar donor. In this review, the typical HMO-producing GTs and the one-pot multienzyme modules for generating various nucleotide sugar donors are introduced, the principles for designing the enzyme cascade routes for HMO synthesis are described, and the important metabolic engineering strategies for mass production of HMOs are also reviewed. In addition, the future research directions in biotechnological production of HMOs were prospected.

Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase.

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

A signature based on glycosyltransferase genes provides a promising tool for the prediction of prognosis and immunotherapy responsiveness in ovarian cancer

BackgroundOvarian cancer (OC) is the most fatal gynaecological malignancy and has a poor prognosis. Glycosylation, the biosynthetic process that depends on specific glycosyltransferases (GTs), has recently attracted increasing importance due to the vital role it plays in cancer. In this study, we aimed to determine whether OC patients could be stratified by glycosyltransferase gene profiles to better predict the prognosis and efficiency of immune checkpoint blockade therapies (ICBs).MethodsWe retrieved transcriptome data across 420 OC and 88 normal tissue samples using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, respectively. An external validation dataset containing 185 OC samples was downloaded from the Gene Expression Omnibus (GEO) database. Knockdown and pathway prediction of B4GALT5 were conducted to investigate the function and mechanism of B4GALT5 in OC proliferation, migration and invasion.ResultsA total of 50 differentially expressed GT genes were identified between OC and normal ovarian tissues. Two clusters were stratified by operating consensus clustering, but no significant prognostic value was observed. By applying the least absolute shrinkage and selection operator (LASSO) Cox regression method, a 6-gene signature was built that classified OC patients in the TCGA cohort into a low- or high-risk group. Patients with high scores had a worse prognosis than those with low scores. This risk signature was further validated in an external GEO dataset. Furthermore, the risk score was an independent risk predictor, and a nomogram was created to improve the accuracy of prognostic classification. Notably, the low-risk OC patients exhibited a higher degree of antitumor immune cell infiltration and a superior response to ICBs. B4GALT5, one of six hub genes, was identified as a regulator of proliferation, migration and invasion in OC.ConclusionTaken together, we established a reliable GT-gene-based signature to predict prognosis, immune status and identify OC patients who would benefit from ICBs. GT genes might be a promising biomarker for OC progression and a potential therapeutic target for OC.

ALG2 inhibits the epithelial-to-mesenchymal transition and stemness of ovarian granulosa cells through the Wnt/β-catenin signaling pathway in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects fertility in women of reproductive age, and a leading cause of anovulatory infertility. Ovarian granulosa cells are a major functional cell type in the ovary that undergo epithelial-to-mesenchymal transition (EMT) to initiate ovulation. Protein glycosylation, catalyzed by specific glycosyltransferases, has been implicated in reproductive events, such as embryo implantation, endometrial receptivity, and decidualization, etc. However, the relationship between glycosylation and EMT-mediated ovulation in PCOS is not well understood. To clarify the role of cobalt chloride (CoCl2) and α-1,3/1,6-mannosyltransferase (ALG2) in PCOS, transwell assay, Real-time PCR, Western blot, immunofluorescence, and sphere formation assay were applied to assess cell migration, invasion, EMT, and stemness of ovarian granulosa cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum level of ALG2 in PCOS patients. We found that CoCl2 promoted the migration, invasion, EMT, and stemness of ovarian granulosa cells by downregulating the expression of ALG2. Upregulation of ALG2 rescued the effects of CoCl2 partially, and inhibited the EMT and stemness of ovarian granulosa cells by inactivating the Wnt/β-catenin signaling pathway. Moreover, the serum level of ALG2 was increased in patients with PCOS. Elevated ALG2, in combination with testosterone, represented better diagnostic value for PCOS as a multimarker than ALG2 or testosterone alone as a single marker. Thus, ALG2, downregulated by CoCl2, was increased in PCOS patients which inhibited the EMT and stemness of ovarian granulosa cells by deactivating the Wnt/β-catenin signaling pathway. The combination of ALG2 and testosterone might thus act as a novel but promising biomarker for PCOS detection.

Discovery of 3'-O-β-Glucosyltubercidin and the Nucleoside Specific Glycosyltransferase AvpGT through Genome Mining.

A genome mining approach was used to identify a hybrid tubercidin-nucleocidin biosynthetic gene cluster (BGC) in Streptomyces sp. AVP053U2. Analysis of culture extracts by liquid chromatography-mass spectrometry revealed the presence of a glucosylated tubercidin derivative. A gene, avpGT, was identified within the hybrid cluster that has homology to the glucosyltransferase that is responsible for 3'-O-β-glucosylation of the fluorinated natural product nucleocidin. AvpGT was heterologously expressed and purified from Escherichia coli for in vitro characterization. AvpGT is active toward UDP-glucose and UDP-galactose as glycosyl donors and several nucleosides as acceptors. Kinetic analysis revealed that AvpGT is most specific for UDP-glucose [kcat/KMapp = (1.1 ± 0.3) × 105 M-1·s-1] as the glycosyl donor and tubercidin [kcat/KMapp = (5.3 ± 1.8) × 104 M-1·s-1] as the glycosyl acceptor. NMR spectroscopic analysis revealed the product of this reaction to be 3'-O-β-glucopyranosyl tubercidin. A sequence analysis of AvpGT reveals a family of nucleoside-specific GTs, which may be used as markers of BGCs that produce glycosylated nucleosides.

Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

Abstract 2400: Fucosyltransferase expression is associated with head and neck cancer survival

Abstract Introduction: Head and neck (H&N) cancers account for 5-7% of new cancer cases in the United States, and most manifest as head and neck squamous cell carcinomas (HNSCC). Fucosyltransferases (FUTs) contribute to the formation of sialofucosylated epitopes, which alter cellular signaling and mediate early steps in metastasis. However, few studies have examined the effect of FUT expression on H&N cancer survival. Here, we sought to examine the association between fucosyltransferase expression and H&N survival and investigate the mechanistic role of fucosylation in H&N cancer. Methods: FUT1-11 expression (mRNA) data was extracted from the Cancer Genome Atlas (TCGA) HNSCC dataset (n=499). Kaplan-Meier, logrank, and Cox proportional hazards tests were conducted. Levels of α1,2-fucosylated epitopes H-antigen, Lewis Y (LeY), and Lewis B (LeB) were measured in normal, dysplastic, and H&N tumor cell lines via flow cytometry, and RT-qPCR was performed to assess FUT expression. To identify fucosylated signaling and adhesion molecules, fucosylated glycopeptides were enriched from CAL27 and HSC-3 tongue squamous cell carcinoma (SCC) lysates via AAL lectin, followed by nLC-MS/MS. EGFR signaling was assessed via western blot, and parallel plate flow assays were performed to assess in vitro metastatic potential, in tumor cell lines with differential FUT expression. Results: In TCGA analyses, the median survival time (MST) of the FUT2 high expression group was 4.7 years, while low expression group MST was 2.7 years, with an adjusted hazard ratio (aHR) of 0.72 (CI = 0.54-0.95). The FUT6-high group MST was 4.7 years versus 2.9 years for low expression (aHR = 0.62, CI = 0.47-0.83). The FUT7-high group MST was 4.7 years versus 3.5 years for low expression (aHR = 0.72, CI = 0.54-0.95). Expression of the LeY epitope, with both α1,2- and α1,3-linked fucose, varied widely in tongue SCC lines, while pharyngeal SCC lines FaDu and Det-562 displayed intermediate LeY levels, via flow cytometry. EGFR, CD44, and integrins, were among the fucosylated glycoproteins identified via mass spectrometry. HSC-3 cells, sorted into low- and high-LeY populations, demonstrated differential EGFR Tyr-1086 phosphorylation, and differences in adhesion to endothelial cells under flow conditions (in vitro), with implications for tumor metastasis. Conclusion: Overexpression of FUT2, FUT6, and FUT7 were associated with statistically significant increases in survivorship. FUT6 and FUT7 are prominently expressed in immune cells, while FUT2 is expressed in endoderm-derived epithelium. Therefore, FUT6 and FUT7 expression may indicate immune cell infiltration into tumor tissue. Future studies will seek to sort tumor tissue based on cell type, to gain an understanding of the cell-type specific glycosyltransferase expression in HNSCC tumors. Our current results will serve as the foundation to interrogate the role of fucosylated glycoproteins in HNSCC metastasis. Citation Format: Kevin Brown Chandler, Brittany Montesino, Nan Hu, Juan M. Lozano, Robert Sackstein. Fucosyltransferase expression is associated with head and neck cancer survival [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 2400.