Dendritic cells (DCs) orchestrate antitumor immunity by integrating signals from the tumor microenvironment to prime effective T cell responses. Many tumors display altered glycosylation patterns, including tumor-associated carbohydrate antigens (TACAs) such as the Tn antigen (GalNAcα1-O-Ser/Thr); yet how these structures influence DC function is not well defined. Here, we investigated how Tn-bearing glycoconjugates modulate DC activation and shape adaptive immunity. Bone marrow-derived DCs (BMDCs) efficiently internalized fluorescently labeled Tn-glycoconjugates, whereas uptake of non-glycosylated counterparts was negligible. Although Tn-glycoconjugates alone did not induce DC maturation, co-stimulation with the Toll-like receptor 9 (TLR9) agonist CpG markedly increased CD86 expression and the secretion of IL-12/23p40 and IL-6, with the multivalent construct MAG:Tn3-PV eliciting the strongest response. These conditioned BMDCs promoted strong IFN-γ production by allogeneic splenocytes, consistent with a Th1-polarizing phenotype. Mechanistically, both uptake and CpG-enhanced activation required the C-type lectin receptor MGL2, as blockade of MGL or competition with GalNAc abrogated glycoconjugate uptake and CpG-enhanced cytokine induction. Pharmacological inhibition revealed that MGL2 signaling synergizes with TLR9 through the Syk-Raf-1-NF-κB axis. In vivo, mice immunized with DCs conditioned with Tn-glycoconjugate- plus CpG displayed enhanced splenocyte proliferation, increased IFN-γ secretion, and elevated cytotoxic activity without IL-10 induction, confirming a Th1-skewed response. Collectively, these findings identify MGL2 as a critical mediator of Tn-glycoconjugate sensing and unveil a synergistic C-type lectin receptor (CLR)-TLR9 cross-talk that amplifies DC maturation and cytotoxic immunity. This study provides mechanistic insight into how specific glycan-lectin interactions fine-tune innate receptor signaling, highlighting the potential of Tn-based glycoconjugates as immunomodulatory tools for vaccine design and cancer immunotherapy.
Extraintestinal pathogenic Escherichia coli O45 is an emerging multidrug-resistant serotype. Herein, we developed a glycoengineered E. coli strain for efficient biosynthesis of O45 O-polysaccharide (OPS45) and the OPS45-based glycoconjugate vaccine. Systematic optimization enhanced the production of OPS45, a polysaccharide containing the rare sugars 6-deoxy-l-talose (6d-l-Tal) and N-acetylfucosamine (FucNAc), with structure confirmed by NMR. Using oligosaccharyltransferase-mediated conjugation in this glyco-optimized chassis strain, we generated a homogeneous cholera toxin B subunit (CTB)-OPS45 glycoconjugate with enhanced antigen loading yield (11.31 ± 0.59 mg/L vs 8.24 ± 0.075 mg/L). LC-MS/MS verified site-specific glycosylation on CTB. Immunization in mice elicited strong O45-specific IgG responses and conferred 90% protection against lethal neonatal meningitis-causing Escherichia coli (NMEC) infection, with a nearly 80% reduction in bacterial burden. These results demonstrate that our integrated biosynthesis and conjugation approach enables rapid and efficient production of a well-defined glycoconjugate vaccine, showing strong potential for combatting resistant NMEC and O45 infections.
Glycoconjugates, glycoproteins, glycolipids, proteoglycans, and glycoconjugate-modifying enzymes play important roles in host-parasite interactions. In this chapter, we focus on the current knowledge of structures and biological roles of glycoconjugates and M60-like metallopeptidases in survival and pathogenesis of the carcinogenic liver fluke, Opisthorchis viverrini. Understanding this chapter could lead to the development of novel molecular tools for detecting, preventing, and treating parasitic O. viverrini infection.
Vaccines are the most effective method for preventing bacterial infections. Identification of the best hapten is an essential step in the development of third-generation Haemophilus influenzae type b (Hib) conjugate vaccines. A comprehensive understanding of the relationship between Hib oligosaccharide chain length and immunological activities remains elusive. A series of Hib capsular polysaccharide (CPS) oligosaccharide derivatives of different lengths were synthesized to establish carbohydrate structure-function relationships. Glycan microarray screening and immunological evaluation of oligosaccharides revealed that tetramer 4, comprising four repeated disaccharide units, is the key immunogenic epitope of CPS. The investigation into the influence of carrier proteins, adjuvants, and vaccination dose on immunogenicity demonstrated that an equivalent dose of glycoconjugate 4-TT without adjuvant can induce a similar immune response as a licensed Hib vaccine (MINHAI Hib). Adjuvant-free 4-TT glycoconjugates can induce both a humoral and a cellular immune response as efficiently as licensed Hib vaccine. This formulation exhibited protective efficacy in a mouse septic infection model through effective pathogen clearance from the circulation to the liver-resident macrophage Kupffer cells and sinusoidal endothelial cells in a mouse septic infection model. In conclusion, a well-defined semisynthetic glycoconjugate vaccine candidate 4-TT was developed with a more straightforward and adaptable manufacturing process and demonstrated excellent efficacy comparable to licensed Hib vaccine, providing a rationale for further evaluation in clinical trials.
Colorectal cancer (CRC) is the third most common cancer globally and a major contributor to cancer-related mortality. Implementation of screening programs in average-risk populations have reduced CRC-related mortality, despite the suboptimal sensitivity of currently used techniques. Novel screening methods with improved accuracy would be expected to further reduce CRC mortality. We previously developed a surface plasmon resonance-based assay that utilizes a N-glycolyl neuraminic acid (Neu5Gc)-specific lectin, SubB2M, to measure levels of Neu5Gc glycoconjugate cancer biomarkers in human serum. In this study, Neu5Gc glycoconjugate levels were measured in the serum of colorectal cancer patients (n = 96) and compared to cancer-free controls (n = 24). A significant increase in serum Neu5Gc levels was observed in patients at every stage of colorectal cancer when compared to the cancer-free control group (P ≤ 0.001). Receiver operating characteristic analysis of these data showed that the assay could distinguish colorectal cancer cases from healthy controls with a sensitivity of 93.75% and specificity of 79.17% and had an AUC of 0.8529. These findings reveal a correlation between serum Neu5Gc and colorectal cancer and highlight the potential for Neu5Gc glycoconjugates to be used as a serum biomarker for colorectal cancer.
Glycoconjugate vaccines are effective against bacterial infections. Key characteristics for optimal immunogenicity are the carbohydrate-to-protein ratio and consequent glycosylation density. Directing the conjugation to specific sites is beneficial when the protein acts as carrier and antigen, however a caveat of this approach is the limited number of conjugated glycans attached which might reduce the anti-glycan immune response. We investigated if polysaccharide length is crucial for optimizing immunogenicity in single-site selective conjugates. We tested conjugates from meningococcal serogroup A and C, and pneumococcal serotype 8 and 14 polysaccharides of varying lengths. Meningococcal protein fHbp was used as carrier. Site-selective conjugates were obtained by thiol-maleimide addition at a C-terminal cysteine, whereas random conjugates were prepared via classic lysine coupling. Results showed that for meningococcal type C and pneumococcal serotypes 8 and 14, oligosaccharides over 15-20 repeating units elicited robust anti-glycan responses, even when attached at a single site. Differently, the less immunogenic meningococcal type A required longer glycan chains for a strong response. Additionally, increasing glycosylation by random conjugation did not enhance polysaccharide immunogenicity but reduced the protein immunogenicity. These findings emphasize that an optimal balance between glycan length and density is essential for effective glycoconjugate vaccine design.
The development of effective vaccines against extraintestinal pathogenic Escherichia coli (ExPEC) serotypes O8 and O9a remains an unmet medical need. We established a sustainable biomanufacturing platform for glycoconjugate vaccines by engineering an E. coli chassis strain with optimized nucleotide sugar precursor supply and enhanced O-polysaccharide (OPS) biosynthesis. Genes related to competitive and catabolic pathways (gnd-rfbB, wcaM-wcaJ and wcaI-wza, glgC, ushA, pfkA/B) were systematically deleted using CRISPR-Cas9 and λ-RED genome editing, while the phosphotransferase system gene ptsA was overexpressed, yielding chassis strain EG01. This rational metabolic engineering increased O8- and O9a-OPS yields by 2.11-fold and 2.4-fold, respectively. Co-expression of Neisseria meningitidis glycosyltransferase PglL and carrier protein CTB enabled efficient protein glycosylation, improving conjugate yields by 2.59-fold (O8) and 4.18-fold (O9a) versus wild-type. Mass spectrometry confirmed site-specific O-glycosylation at CTB Thr19 with preserved OPS repeat-unit structure. The vaccine candidate demonstrated a favorable safety profile in mice, inducing only transient cytokine responses without systemic toxicity. Immunization elicited robust, durable Th1-biased IgG responses, conferring 80-90% protection against lethal challenge and significantly reducing bacterial loads in vivo. This work establishes an efficient, scalable glycoconjugate vaccine production platform, highlighting a promising metabolic engineering strategy to combat ExPEC infections.
Pseudomonas aeruginosa is an antibiotic-resistant pathogen and leading cause of hospital-acquired infections. Its ability to form biofilms enables persistent colonization by protecting bacterial cells from antibiotics and host immune action. The Pel exopolysaccharide is a key component of the biofilm, contributing to intercellular adhesion and structural integrity. Although Pel has been implicated in P. aeruginosa pathogenesis, its immunogenic potential remains underexplored. In this study, a panel of synthetic Pel-derived oligosaccharides was prepared with a thiol-functionalized linker to enable site-selective conjugation to the carrier protein CRM197. The resulting glycoconjugates were administered to mice and elicited robust antibody responses. Among these, one hexasaccharide conjugate induced antibodies that bound strongly to both the natural Pel polysaccharide and biofilm forming P. aeruginosa cells. These findings support the feasibility of using well-defined synthetic Pel-structures in glycoconjugate vaccine development and provide a molecular framework for targeting biofilm-associated antigens in P. aeruginosa.
Klebsiella pneumoniae (KP) is a critical gram-negative opportunistic pathogen, causing not only nosocomial infections that can be antibiotic resistant but also leading to pyogenic liver abscess (PLA) disease. Armed with knowledge of the chemical structures of K. pneumoniae K1 and K2 surface capsular polysaccharides (CPSs) and their CPS depolymerases from bacteriophages, we developed potential glycoconjugate vaccines against this organism. This was done by using CPS polymerases present in tail spike proteins (TSPs) of specific KP bacteriophages to cleave the CPS and then chemically conjugating the fragment oligosaccharides to carrier proteins.
Polysaccharide-protein conjugate vaccines have proven highly effective, yet they remain limited by manufacturing complexity, cost, and variable performance across serotypes, while carrier proteins can add unwanted immunological and production burdens. To address these constraints, we explored a carrier-protein-free conjugate vaccine concept in which a broadly MHC class II-binding helper epitope (PADRE) replaces the conventional protein carrier to provide T-cell help for a pneumococcal capsular polysaccharide antigen. Using serotype 15C CPS as a model, we generated CPS-PADRE conjugates and compared designs with or without a putative cleavable motif (RR) at the junction, alongside a conventional protein conjugate as a benchmark. In mice, the CPS-protein conjugate induced the strongest CPS-specific IgG response, whereas CPS-PADRE conjugates elicited clear but overall lower antibody levels. Notably, incorporation of the cleavable motif did not improve immunogenicity and instead reduced humoral responses relative to the non-cleavable design. These findings support the feasibility of carrier-protein-free polysaccharide-peptide conjugate vaccines, while highlighting that cleavable junctions are not universally advantageous and must be empirically optimized for polysaccharide-helper epitope architectures.
Sialic acids (Sia) on glycoproteins and glycosphingolipids act as receptors for many respiratory viruses, including human influenza A viruses and respiroviruses, which typically bind to α2-6- and α2-3-linked Sia (2-6Sia and 2-3Sia), respectively. In view of this binding discrepancy and as the exact sialoglycoconjugates supporting respirovirus infection remain unclear, we assessed the receptor requirements for infection and binding of several respiroviruses and avian Newcastle disease virus (NDV). To this end, we employed a library of isogenic HEK293 cells displaying defined sialoglycoconjugates. Respirovirus infection was shown to strictly depend on 2-3Sia, with a preference for the terminal Siaα2-3Galβ1-4GlcNAc sialoglycotope regardless of the underlying glycoconjugate structure, whereas NDV showed a broader sialoglycotope usage. Notably, 2-6Sia enhanced respirovirus infection in low 2-3Sia contexts via heteromultivalent surface binding, which resulted in prolonged virus-receptor interactions. This heteromultivalent binding is expected to aid respirovirus infection of the 2-6Sia-rich human upper respiratory tract.
Bone tuberculosis is characterized by severe bone destruction driven by aberrant osteoclast overactivation. However, the direct mechanism by which Mycobacterium tuberculosis (Mtb) mediates this pathological process remains unclear. Understanding the molecular basis of pathogen-driven osteoclast dysregulation is essential for developing effective host-directed therapeutic strategies. Transcriptomic profiling was performed to identify differentially expressed sialylation-related genes and activated signaling pathways in Mtb-infected cells. Murine bone-tuberculosis models and in vitro osteoclast cultures were employed to assess osteoclast activity and surface α2,3-sialylation levels following Mtb infection. Functional interventions included enzymatic removal of α2,3-sialic acid and pharmacological inhibition of ST3GAL1. Metabolomic analysis was conducted to characterize Mtb-induced alterations in glycerophospholipid metabolism. Transcriptomic profiling revealed upregulation of sialylation-related genes and activation of TLR2-dependent signaling upon Mtb infection, providing a molecular basis for pathogen-driven surface glycan modifications. In both murine bone-tuberculosis models and in vitro osteoclast cultures, Mtb infection concurrently enhanced osteoclast activity and surface α2,3-sialylation. Enzymatic desialylation or ST3GAL1 inhibition markedly attenuated this overactivation. Metabolomic analysis further demonstrated Mtb-induced reprogramming of glycerophospholipid metabolism, potentially supplying substrates for sialylated glycoconjugate biosynthesis. These findings identify α2,3-sialylation as a central driver of Mtb-induced pathological osteoclast activity, mechanistically linking TLR2 signaling, surface glycan remodeling, and metabolic reprogramming. The coordinate regulation of membrane glycoconjugate biosynthesis and glycerophospholipid metabolism suggests an integrated host response exploited by Mtb to promote bone destruction. Collectively, host glycosylation machinery and associated metabolic pathways represent promising targets for host-directed therapy in bone tuberculosis.
In recent years, natural proteins have increasingly failed to meet modern food industry demands for multifunctional performance under advanced processing conditions. Glycosylation, as an eco-friendly and safe protein modification strategy, has emerged to improve protein functional characteristics. This review systematically summarizes three predominant glycosylation approaches, that is, Maillard reaction-based glycation, enzymatic glycosylation, and physically-assisted glycosylation techniques. Furthermore, the study comprehensively illustrates process characteristics of dry-heating, wet-heating, enzymatic, and physically-assisted methods in glycoconjugate preparation. Moreover, critical reaction parameters are emphasized, including saccharide type selection, thermal processing conditions protein-to-carbohydrate mass ratios, pH, and temporal control. Comparative analyses demonstrate that glycated proteins exhibit superior physicochemical properties versus native counterparts: enhanced solubility, optimized emulsifying activity, strengthened gelation capacity, and improved foaming stability. Besides, industrial applications are multifaceted. Glycosylated proteins can serve as emulsifiers or gelling agents for the preparation of emulsion/gel systems. These systems function as fat replacers in baked goods and meat products, encapsulate bioactive compounds for the development of functional foods, and demonstrate emerging potential in 3D food printing by improving rheological properties. Notably, glycoconjugates exhibit intrinsic antimicrobial effects and allergenicity reduction properties, significantly expanding their commercial viability and safety profile in food applications. Finally, this review critically analyzes the potential health risks associated with protein glycosylation and proposes potential solutions. It provides critical insights into the structure-function relationships of glycosylated proteins and provides a summary of the inherent limitations of this process. These comprehensive discussions establish a conceptual framework that paves the way for future developments in food technology trends.
Helicobacter pylori is a Gram-negative pathogen that persistently inhabits the human gastric mucosa and infects over half of the world's population.The lipopolysaccharide (LPS) of H. pylori represents a promising target for developing effective glycoconjugate vaccines. Herein, we report the first chemical synthesis of the LPS-derived pentasaccharide fragment from H. pylori, accomplished via a linear [(1+(1+(1+(1+1))))] glycosylation strategy. High stereoselectivity in the formation of 1,2-cis-L-fucosyl and 1,2-cis-D-glucosyl linkages was achieved through remote participation effects of the O4-benzoyl and O6-levulinoyl protecting groups, respectively. N-Phenyl trifluoroacetimidate glycosyl donors demonstrated higher efficiency than thioglycoside donors in glycosylation reactions, while TBSOTf proved superior to TMSOTf as a promoter for low-reactivity hydroxyl acceptors. In addition, a series of di-, tri-, and tetrasaccharide fragments bearing an aminopropyl linker were synthesized, facilitating glycan microarray construction and in vivo immunological evaluation, and thereby accelerating the development of a synthetic glycoconjugate vaccine against H. pylori.
Invasive fungal infections caused by Candida albicans pose a serious threat to human health, particularly in immunocompromised individuals. Surface-exposed β-glucans of C. albicans have been identified as promising targets for antifungal vaccine development. However, conventional glycoconjugate vaccines often suffer from limited immunogenicity. Herein, we report an innovative vaccine strategy that recruits endogenous antibodies, such as anti-rhamnose (Rha) antibodies, to improve antigen delivery to antigen-presenting cells (APCs). A redox-responsive disulfide linker was incorporated into the vaccine design to promote endosomal escape and enhance antigen processing. The construct was prepared by conjugating β-glucans to bovine serum albumin (BSA), followed by coupling Rha haptens via the redox-responsive linker, yielding β-glucans-BSA-L2-Rha. Immunological evaluations demonstrated that the presence of endogenous anti-Rha antibodies substantially enhanced immune responses, with the redox-responsive glycoconjugate eliciting the strongest reactivity. Moreover, antisera generated by the vaccine effectively recognized the hyphal form of heat-killed C. albicans (HKCA). This study establishes a broadly applicable strategy for the further development of antifungal vaccines.
This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel®, AH; Adju-phos®, AP; and CpG-1018®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2-8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing.
Tumor-associated glycosylation is a defining hallmark of cancer, exerting profound effects on multiple aspects of tumor biology. This phenomenon arises from the central role of glycosylation in a wide range of cellular processes and its inherently diverse structural complexity. In cancer cells, aberrant glycosylation often results in the modification of glycoconjugate structures, leading to alterations in cell surface architecture that disrupt cellular homeostasis and signaling pathways. These changes can enhance tumor cell proliferation, invasion, and metastasis by modulating cell adhesion, receptor activation, and intracellular communication. Beyond its direct impact on cancer cells, tumor-associated glycosylation plays a pivotal role in shaping the tumor microenvironment. Aberrant glycan structures influence immune cell infiltration by altering antigen presentation and immune checkpoint interactions, contributing to immune evasion. Additionally, these modifications regulate angiogenesis by affecting endothelial cell function and promoting the formation of aberrant vasculature, which supports tumor growth and metastasis. Glycosylation also mediates tumor-stroma interactions, influencing extracellular matrix remodeling and fibroblast activation, further enhancing cancer progression. This interplay between cancer-associated glycan modifications and their functional roles in tumorigenesis presents a promising therapeutic approach. Unlike conventional treatments, glycan-targeting therapies confer high tumor specificity, operate independently of canonical immune checkpoint targets, and help mitigate immune cell exhaustion. This review explores commonly dysregulated glycan motifs implicated in tumorigenesis and delves into their mechanistic contributions to cancer pathogenesis. We then highlight emerging opportunities for therapeutic intervention, including the development of glycan-targeted therapies and biomarker-driven strategies for cancer diagnosis and treatment. We also outline where glycan-targeted agents (e.g., desialylating biologics, glycomimetics, and anti-glycan mAbs) can complement checkpoint blockade and potentially overcome immune escape.
Streptococcus agalactiae or Group B Streptococcus (GBS) is a major causative agent of neonatal sepsis, meningitis, still births and postnatal complications in women. Capsular polysaccharides (CPS) on the bacterial surface are implicated in pathogen virulence by evasion of host cellular defense mechanisms and have in turn served as attractive vaccine candidates for maternal immunization. In this study, we probed the role of a key structural feature, N-acetylation of constituent sugars in GBS capsular polysaccharide, on product characteristics and preclinical immunogenicity. Loss of N-acetyl groups in CPS during the purification stage requires a re-N-acetylation step to restore the N-acetylation levels in the polysaccharide. GBS serotype (ST) III CPS with different % N-acetylation levels were prepared and utilized for conjugate vaccine preparation involving rCRM197 as a carrier protein and subjected to structural and analytical characterization and immunogenicity evaluation in rabbits. Although different levels of re-N-acetylation in CPS as determined by proton NMR did not impact glycoconjugate stability, serological analysis revealed strong and direct correlation of N-acetylation levels in the CPS to the quality of neutralizing (IgG) and functional antibody responses to the GBS III vaccine. Collectively, the study findings suggest N-acetylation to be a salient structural feature and part of the immunodominant epitope in GBS CPS that defines the critical product attributes dictating vaccine quality and immunogenicity.
Chemical communication is essential for mammalian survival from the earliest stages of life, yet most of what is known about the prenatal development of the olfactory and vomeronasal systems comes from laboratory rodents. Here we investigated the fetal development of the nasal chemosensory systems in the fossorial water vole (Arvicola scherman), a free-living arvicoline rodent with a subterranean lifestyle. We analyzed fetuses at embryonic days E17 and E21 (term) using classical histology, immunohistochemistry and lectin histochemistry. This approach enabled to assess structural maturation, neuronal differentiation, and the temporal dynamics of glycoconjugate expression in the vomeronasal organ (VNO), olfactory epithelium, and the main (MOB) and accessory olfactory bulbs (AOB). By E21, the MOB displayed a layered adult-like organization with well-defined glomeruli and interneuronal populations, whereas the AOB showed delayed morphological maturation but already exhibited selective molecular signatures in its superficial layers. Prenatally, the VNO underwent conspicuous structural differentiation, including stratification of the sensory epithelium, axonal fasciculation, and development of vomeronasal glands. Immunohistochemical analysis revealed early expression of G-protein subunits and calcium-binding proteins, indicating premature pathway specification and interneuronal circuit formation. Lectin labeling provided additional insights: SBA emerged as a selective marker of the vomeronasal pathway; UEA characterized early compartmentalization of vomeronasal projections and LEA showed a pan-chemosensory binding pattern. Together, these findings demonstrate that Arvicola scherman exhibits a remarkably accelerated prenatal maturation of its chemosensory systems compared with laboratory rodents. This early functional readiness emphasizes the importance of incorporating wild species into developmental neurobiology to refine our understanding of chemosensory evolution.
Cytotoxic CD8+ T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8+ T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8+ T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8+ T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8+ T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8+ T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8+ T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8+ T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.