Modern glycobiology is a result of multiple revolutionary and ground-breaking observations that both challenged and eventually changed prior perspectives about the expression and biological roles of glycoconjugates. These sweeping transformations in the field altered concepts that were embedded in what may be termed paradigms. Some prior paradigms included ideas that are still echoed in the current scientific literature, such as glycosylation of proteins is only found on secreted and membrane-associated proteins; glycans do not have clear and specific functions; protein binding to carbohydrates is relatively low affinity and non-specific; and protein glycosylation is relatively random and highly heterogeneous. As prior paradigms reached a crisis in terms of their supportability, they were replaced by new paradigms. The number of such new paradigms is large and documenting them is important. These modern paradigms include the idea that wide-spread modifications of amino acids with different sugars are common, that specific protein glycosylation occurs, the presence of glycoproteins in all cellular compartments, numerous roles of glycans and genes encoding their synthesis in human diseases, and the nature of carbohydrate recognition beyond simple sugars, and many others. Many researchers may not be aware of these emerging paradigms that have been developing in the research community since the 1960s. Here we summarize these new paradigms and some are discussed in detail from an historical perspective. These new paradigms demonstrate the essentiality and novelty of glycans and their biological functions.
Protein O-mannosylation is an evolutionarily conserved post-translational modification essential for neuromuscular development and physiology. The obligate heteromeric complex of protein O-mannosyltransferase 1 and 2 (POMT1/2) initiates O-mannosylation of α-dystroglycan and a limited number of known additional substrates in the endoplasmic reticulum. In humans, defects in these enzymes disrupt dystroglycan function, leading to impaired extracellular matrix interactions and causing a spectrum of severe congenital muscular dystrophies termed dystroglycanopathies. However, mounting evidence indicates that POMTs have additional functional substrates that substantially contribute to POMT1/2 mutant phenotypes through dystroglycan-independent mechanisms. This review examines POMT1/2 function through a comparative evolutionary lens of model organisms, such as Drosophila and zebrafish, and discusses O-mannosylation substrates beyond dystroglycan. We review the functions of POMT1/2 in muscles and the nervous system in Drosophila, including the interplay with receptor protein tyrosine phosphatases, a novel conserved family of substrates with essential functions in neural circuit formation. In the zebrafish model, POMT1/2 mutant phenotypes recapitulate multi-tissue defects associated with dystroglycanopathies, revealing compensatory mechanisms with broad implications for understanding these diseases and also suggesting the involvement of dystroglycan-independent pathomechanisms. Together, these animal models provide a tiered experimental framework, combining the advantages of simplified glycan architectures, powerful genetic tools, and comprehensively characterized development, with evolutionary conservation of core molecular players involved in human disease. These and other animal models will continue to make invaluable contributions towards dissecting POMT1/2 function at molecular, cellular, and genetic levels, leading to a better understanding of the pathological mechanisms of dystroglycanopathies.
Insulin resistance is a major complication of obesity. In adults with obesity insulin resistance is associated with a hyposialylation of the Fc-linked glycan on IgG, and a causal link between IgG hyposialylation and glucose dysregulation has been demonstrated in obese mice. What is unknown is how obesity causes the changes in IgG glycosylation. To avoid factors besides obesity that may influence IgG glycosylation, we sought to fill this knowledge gap by studying adolescents with and without obesity. We demonstrate that in pediatric obesity IgG is hyposialylated and hypogalactosylated, with the changes most apparent in females, and that this is related to an upregulation of B cell WNT3, a GWAS-identified candidate gene for IgG glycosylation whose function in glycan modulation was previously unknown. In parallel, WNT3 is upregulated in B cells from obese mice. Linkage between WNT3 upregulation and decreased IgG galactosylation and sialylation was demonstrated in both a HEK293FS cell model and an ARH-77 B lymphoblast cell line. These observations indicate that obesity causes IgG hypogalactosylation and resulting hyposialylation by previously unrecognized actions of WNT3 which regulate IgG galactosylation in B cells. Better understanding of how obesity alters the unique glycobiology of IgG offers the possibility of identifying additional therapeutic targets in the battle against the insulin resistance that complicates obesity.
The lactating mammary gland is uniquely adapted to synthesize unconjugated free glycans known as human milk oligosaccharides (HMOs). HMO biosynthesis is initiated by the formation of lactose, the common precursor from which structurally diverse neutral and acidic oligosaccharides are generated through the sequential action of Golgi-resident glycosyltransferases. Although microbial fermentation and chemical synthesis have enabled production of selected HMOs, human cell-based platforms remain largely unexplored despite providing the native glycosylation machinery required for HMO assembly. Consequently, the contributions of individual glycosyltransferases to HMO biosynthesis have remained difficult to define. Here, we describe the reconstruction of HMO biosynthesis in human embryonic kidney (HEK293) cells through reconstitution of the lactose synthase complex and systematic engineering of downstream glycosyltransferases. This modular approach enables programmable production of structurally defined HMOs, including both simple sialylated oligosaccharides and more complex type-I and type-II structures, while providing a tractable platform for assigning glycosyltransferase functions within the HMO biosynthetic pathway. Together, this establishes mammalian glycoengineering as a versatile system for investigating HMO biosynthesis and for the tailored production of biologically relevant HMOs.
To investigate the roles of the α2,3-sialyltransferase ST3Gal1 and the α2,6-sialyltransferase ST6Gal1 in T cell-mediated tumor control, we generated mice with mature T cell-specific deletion of ST3Gal1 (T-ST3KO) or ST6Gal1 (T-ST6KO) using distal Lck-Cre-mediated recombination. Deletion of ST3Gal1 in mature T cells did not affect thymic T cell development but resulted in a reduction of peripheral CD8+ T cells. In contrast, ST6Gal1 deletion had minimal impact on T cell development and peripheral T cell abundance. Following in vitro stimulation of isolated T cells from T-ST3KO and parental wild type (WT) mice with anti-CD3 plus anti-CD28/CD80-Fc, CD8+ T cells from T-ST3KO mice exhibited enhanced activation and an increased frequency of CD44 positive memory-like T cells, while comparison of T cells from T-ST6KO and the parental WT mice showed no significant changes. Despite the activated CD8+ T cell phenotype in T-ST3KO mice, subcutaneous MC38 tumors displayed accelerated growth. In contrast, tumor progression in T-ST6KO mice was unchanged from the parental WT mice. Notably, T-ST6KO mice developed increased pulmonary metastases following intravenous challenge with B16F10 melanoma cells, whereas metastatic burden was unaffected in T-ST3KO mice. These findings demonstrate distinct and non-redundant roles for ST3Gal1- and ST6Gal1-mediated sialylation in regulating T cell function and antitumor immunity and reveal context-dependent effects of T cell intrinsic sialylation in controlling primary tumor growth and metastatic dissemination.
The Human Cytomegalovirus (HCMV) gH/gL/UL128/UL130/UL131A complex (Pentamer) is among the candidates for the development of a vaccine against HCMV; indeed, it brought to more immunogenic formulations which were able to protect the fetus against vertical transmission in pregnant women. Pentamer-specific antibodies have shown to be hundredfold more potent than gB or gH/gL antibodies, endowed with strong neutralizing activity. The often inadequate antibody immune response elicited by glycoprotein antigen has been, in part, attributed to the abundance of glycosylation on the protein. Here, we determine the composite glycan population of each of the N-linked and O-linked glycosylation sites present on the Pentamer by multienzymatic proteolysis and mass spectrometry; furthermore, total N-glycans profiles of Pentamer expressed in different cell systems have been compared exploiting the glycan fluorescent labeling and the immunogenicity of Pentamer with different glycosylation patterns has been investigated. Our analysis reveals the presence of underprocessed oligomannose-type glycans on some glycosylation sequons, which are probably derived as a result of sterically reduced accessibility to glycan processing enzymes. Furthermore, the antigen glycosylation pattern affects the elicitation of neutralizing antibodies in mice.
The factors governing protein-specific N-glycosylation remain incompletely understood despite extensive knowledge of the biosynthetic machinery involved in glycan assembly. In particular, the contribution of the protein sequence itself to the final glycosylation pattern has received comparatively little attention. Here, we employed isotope-assisted NMR spectroscopy to investigate the N-glycosylation of the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD-SCoV2) produced in HEK293F cells. Previous analyses revealed the unexpected presence of terminal GalNAc-containing epitopes, including LacdiNAc (LDN), sulfated LacdiNAc (4S-LDN), sialylated LacdiNAc (6Sia-LDN), and fucosylated LacdiNAc (LDNF). Inspired by earlier studies showing that certain β4GalNAc-transferases recognize basic peptide motifs, we generated an RBD variant carrying four substitutions of solvent-exposed basic residues located near the N-glycosylation sites. Comparative NMR analysis demonstrated that the mutant protein exhibits a marked reduction in GalNAc-containing terminal epitopes, accompanied by a corresponding increase in Gal-containing structures. The overall protein fold remained unchanged, indicating that the observed glycosylation differences arise from altered glycan processing rather than major structural perturbations. These findings provide direct evidence that local protein sequence features modulate the incorporation of terminal glycan epitopes during Golgi processing. More broadly, this work highlights the power of quantitative NMR spectroscopy for the structural characterization of glycoproteins and for detecting subtle changes in glycan populations that are directly linked to protein sequence.
α-1,2-glucans are polysaccharides characterized by their unique α-1,2-glycosidic bonds and right-handed helical structure, which suggest novel biological functions. The alr1000 protein in Nostoc sp. PCC 7120 is the key enzyme for synthesizing α-1,2-glucans. In this study, we prepared rabbit antiserum for alr1000 and investigated its expressions when Nostoc sp. PCC 7120 is subjected to high salinity and high temperature stress. The results showed that under high salinity (up to 200 mM) and high temperature (40 °C) conditions, alr1000 expression reached its highest level at 4 h. This suggests that the elevated expression of alr1000 initiates massive production of α-1,2-glucans within Nostoc sp. PCC 7120, which helps the organism survive these stressful conditions. In addition, we identified and characterized the degrading enzymes of α-1,2-glucans. All4989, a GH65 family α-1,2-glucan phosphorylase from Nostoc sp. PCC 7120, was found to degrade α-1,2-glucans through a mechanism where a phosphate serves as the nucleophile to cleave the α-1,2-glycosidic bonds of α-1,2-glucans, while Glu485 functions as a general acid. Furthermore, the enzyme also catalyzes an efficient reverse reaction, synthesizing α-1,2-glucans of different molecular weights using breakdown products as substrates. Mechanistically, this process involves a key functional shift: Glu485 acts as a general acid during the forward degradation and switches to acting as a general base during the reverse synthesis.
The endothelial glycocalyx plays a pivotal role in maintaining blood-brain barrier integrity, and its degradation promotes leukocyte adhesion and transendothelial migration. We previously reported that the degradation of heparan sulfate (HS), a major structural component of the glycocalyx, aggravated inflammation after stroke onset. In addition, acrolein (ACR), an α,β-unsaturated aldehyde generated following stroke onset, promoted HS degradation via activation of proheparanase-1 (proHPSE1), the inactive precursor of heparanase-1 (HPSE1). However, whether this ACR-mediated HS degradation occurs in a cell-type specific manner and contributes to endothelial glycocalyx dysfunction, particularly by enhancing leukocyte adhesion, remains unclear. In this study, we demonstrated that ACR-mediated HS degradation promotes THP-1 cell adhesion to human immortalized brain microvascular endothelial cells (HBMEC/ci18). Immunocytochemistry staining indicated that proHPSE1 was visualized on both HBMEC/ci18 and EA.hy926 cells. However, HS localization in HBMEC/ci18 cells differed from that in EA.hy926 cells, which may underlie the differential response of ACR-dependent HS degradation. Furthermore, supplementation of proHPSE1 containing ACR-carbonylated lysine residues directly induced HS degradation in HBMEC/ci18 cells. These results suggest that monocyte adhesion following glycocalyx degradation may be triggered by ACR-induced carbonylation of proHPSE1 during brain infarction. HBMEC/ci18 cells exposed to ACR or treated with carbonylated proHPSE1 provide a useful model for screening agents that protect the endothelial glycocalyx.
Cell-surface glycans play essential roles in cell communication, immune recognition, and disease progression. Their medical applications are supported by two important technological pillars: glycan recognition and glycan editing. This Review focuses on three key directions: recognition technologies, namely, in situ imaging and glycomic profiling, for detecting and profiling glycans; functional editing technologies, including genetic, enzymatic, chemical, and metabolic approaches, for precisely remodelling glycans; and medical applications driven by the synergy between recognition and editing, with a focus on four areas: biomarkers, lectin-based therapies, precision glycan editing, and cancer immunotherapy. Throughout the Review, five representative glycan classes-high-mannose N-glycans, mucin-type O-GalNAc, heparan sulfate, ganglioside GM3, and glycoRNAs-are used as recurring examples wherever possible. This Review offers an integrated perspective to navigate the process by which glycan recognition and editing technologies collectively drive the translation of cell-surface glycan research into clinical practice.
Aberrant O-glycosylation and miRNA dysregulation are established features of tumor aggressiveness, particularly in triple-negative breast cancer (TNBC). Using a Cosmc-silenced 4T1 murine model (Tn+), characterized by truncated O-glycans and enhanced metastatic potential, tissue and serum miRNA profiling identified miR-21-5p as a circulating marker associated with the Tn+ phenotype. Transcriptomic analysis revealed that miR-21-5p targets multiple tumor suppressor genes, including Btg2, Spry1, Tbx2, Rhob, and Dusp8, suggesting its involvement in epithelial-mesenchymal transition and immune modulation. Integrative single-cell RNA sequencing (scRNA-seq) of the 4T1 tumor microenvironment revealed distinct cellular clusters with inverse expression patterns of GALNT enzymes (involved in Tn synthesis) and miR-21-5p target genes, defining Tn+-like and Tn--like subpopulations. Translating these findings to human breast cancer (TCGA-BRCA), a prognostic model combining clinical variables (age, metastasis, PAM50 subtype) with three-gene expression (OLR1, PCSK6, and GALNT6) significantly improved patient risk stratification (P = 0.015). By integrating multi-omics analyses (scRNA-seq, TCGA) with an aggressive Tn+ TNBC model, this study defines a novel three-gene prognostic signature that links the glyco-miRNA axis to tumor aggressiveness, offering a promising tool for advanced patient stratification and the development of precision glyco-therapeutics.
Aberrant O-GalNAc glycans such as Tn, STn, and T are among the most consistent tumor-associated carbohydrate antigens, broadly expressed on carcinomas but largely absent from healthy epithelia. In parallel, O-glycans can also be modified to carry functional motifs, such as Lewis antigens. Rather than a simple shift from elongated to truncated structures, cancer-associated O-glycans form a heterogeneous repertoire of truncated and elongated glycoforms that coexist across the tumor glycocalyx. Collectively, both short and elongated cancer-associated O-glycans co-drive tumor formation through their simultaneous influence on multiple cancer hallmarks, including adhesion, receptor signaling, dissemination, and immune evasion. The latter occurs through interactions with glycan-binding proteins including Selectins, Siglecs, macrophage galactose-type lectin (MGL), and galectins. The stable expression of especially truncated O-glycans across cancer stages, as well as their driving influence on cancer progression, make short, truncated O-glycans attractive therapeutic targets. While early vaccine approaches had limited efficacy, strategies that couple O-glycan recognition with potent effector mechanisms have shown promise. This includes O-glycan-directed chimeric antigen receptor T cells (CARTs), T-cell bispecifics (TCBs), and particularly antibody-drug conjugates (ADCs), which have demonstrated strong preclinical activity. Looking forward, multi-specific antibodies, bio-orthogonal chemistry, and artificial intelligence-driven engineering are expected to enhance safety, selectivity, and improve patient stratification, helping to further exploit O-GalNAc glycans in precision oncology.
Human milk oligosaccharides (HMOs) are unconjugated and structurally diverse glycans synthesized in the lactating mammary gland through the stepwise action of glycosyltransferases that extend a free lactose core. Several HMOs are capped with sialic acids, including 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), that promote early-life microbiota development and contribute to immune system and neuronal functions. These health-promoting properties make sialylated HMOs attractive biomolecules for incorporation in infant nutrition and functional food products. Mammalian cell lines lack endogenous HMO production, limiting mechanistic studies of HMO biosynthesis and constraining production strategies based on human cells. Here, we developed a human cell-based strategy for the production of the two common sialyllactose isomers 3'-SL and 6'-SL in glycoengineered human embryonic kidney (HEK293) cells. We co-expressed LALBA and B4GALT1, that together form the lactose synthase complex, to introduce free lactose biosynthesis capacity into a genetically engineered human cell line without sialylation (HEK293ΔSia). Stable expression of either ST3GAL or ST6GAL isoenzymes in HEK293ΔSia cells revealed that ST3GAL3/4/5, and especially ST3GAL5, efficiently convert lactose into 3'-SL while ST6GAL1 and ST6GAL2 produce the 6'-SL isomer. These results provide insights into the in vivo ability of sialyltransferase isoenzymes to use lactose as substrate. Establishing HMOs biosynthesis pathways into controllable human cell systems offers an alternative strategy for production of HMOs and provides a starting point to unlock biosynthesis of more complex HMOs in human cells.
Siglec-15 has emerged as a therapeutic target in cancer, yet the glycan determinants and protein scaffolds that mediate engagement between Siglec-15-expressing myeloid cells and tumor cells remain incompletely defined. Here, we investigated the molecular basis of Siglec-15 recognition of cancer cells and examined transcriptional as well as functional programs associated with Siglec-15 expression in tumor-associated myeloid populations. Using immunoprecipitation-mass spectrometry in the pancreatic cancer cell line AsPC-1, we identified multiple mucin-domain glycoproteins enriched in Siglec-15 pulldowns. Additionally, disruption of glycan structures demonstrated that both complex N-glycans and extended mucin-type O-glycans contribute to optimal Siglec-15 binding. To define the myeloid population associated with Siglec-15 in human tumors, we interrogated publicly available single-cell RNA sequencing datasets and found that SIGLEC15 expression is enriched within a subset of tumor-associated myeloid cells exhibiting transcriptional features linked to osteoclast differentiation and extracellular matrix remodeling. Finally, in a THP-1 coculture model, Siglec-15 was further associated with DAP12-dependent tumor-induced expression of the osteoclast markers ACP5 and MMP9, together with increased release of IL-1β and IL-6. Collectively, these findings identify glycan and glycoprotein features that support Siglec-15 binding to malignant cells and associate SIGLEC15 expression with osteoclast-like and matrix remodeling myeloid programs in human cancers, providing a framework for mechanistic studies of this glyco-immune checkpoint.
The N-linked tetrasaccharide decorating glycoproteins of the halophilic archaea Halobacterium salinarum offered the first example of N-glycosylation outside the Eukarya and still represents the only known instance of iduronic acid (IdoA) being employed in this post-translational modification. Recent identification of Agl32 as the D-glucuronyl C5-epimerase catalyzing the conversion of glucuronic acid (GlcA) into IdoA allows for comparing this enzyme in each of the three domains of life, namely, Eukarya, Bacteria and Archaea. Specifically, the current study assessed whether Agl32 requires flanking sugars on either side of the target GlcA, as do its eukaryal and bacterial counterparts. Nuclear magnetic resonance analysis of the glycan from an Hbt. salinarum mutant unable to add the fourth and final N-linked tetrasaccharide sugar revealed that Agl32 requires GlcA on both sides of the target GlcA for the epimerization reaction. Despite similar requirements for flanking sugars, Agl32 shares little structural similarity with eukaryal GlcE or DSepi1, D-glucuronyl C5-epimerases respectively involved in IdoA generation in the glycosaminoglycans heparin/heparan sulfate and dermatan sulfate. Moreover, Agl32 processes a substrate far shorter than what is recognized by the eukaryal enzymes. Indeed, it would appear that Agl32 relies on a catalytic mechanism distinct from that employed by these other D-glucuronyl C5-epimerases. Finally, the presence of agl32 homologues in putative N-glycosylation gene clusters in other haloarchaea argues that the use of IdoA in N-glycosylation extends beyond Hbt. salinarum.
Glycan sialylation is vital for proper cellular function and signaling. The six-membered ST3GAL family of sialyltransferases catalyzes the transfer of sialic acid in an α2,3-linkage to galactose residues on the outermost glycan epitopes. Dysregulation of sialyltransferase activity has been linked to diverse pathological processes. To expand our understanding of the substrate specificity and cooperative function of the ST3GAL family enzymes in protein and lipid glycosylation, we systematically analyzed the function of individual ST3GAL enzymes in glycan biosynthesis using a panel of CRISPR/Cas9-engineered human keratinocyte (N/TERT-1) cell lines with single or combined ST3GAL gene knockouts (KO). For protein glycosylation, KO of ST3GAL1 reduced sialylation of type 3 epitopes (Galβ1,3-GalNAc-) on both core 1 and 2 O-glycans, while complete ablation of sialylation was observed for the combined ST3GAL1 and ST3GAL2 KO. ST3GAL2 KO alone had limited effect, but reduced sialylation of specifically core 1 O-glycans. KO of ST3GAL4 and the combined KO of ST3GAL4 and ST3GAL6 reduced sialylation of type 1 and 2 epitopes (Galβ1,3/4-GlcNAc-) on both N- and O-glycans, while no effect was observed for the single KO of ST3GAL6. In GSL glycan biosynthesis, ST3GAL5 regulated lactosylceramide sialylation, as anticipated. ST3GAL2 and ST3GAL6 mediate sialylation of type 3 motifs, whereas ST3GAL3 and ST3GAL6 target type 2 epitopes, with ST3GAL3 exhibiting no discernible preference between type 1 and type 2 substrates. Our findings reveal that glycosyltransferase specificities are shaped by substrate availability, epitope distribution across glycan classes, and enzyme competition, which can only be captured by investigating this within the cellular context.
Increased sialylation of tumour cells, which favours tumour growth and immune evasion, has been described using in vitro and in vivo models, leading to the first in-human clinical trial targeting sialylation in cancer. One important limitation is that the biology of sialic acids and their receptors (Siglecs), which have been considered as immune checkpoints, is different between mice and human. Hence, it is crucial to fully describe and investigate sialic acids and Siglecs expression in animal models, to define their advantages and limitations. Here, we determined the sialylation profile of the widely-used B16OVA melanoma mouse model using flow cytometry and glycomics. B16OVA cells express Siglec-E-binding sialoglycans, therefore we explored Siglec-E expression across various tissues from tumour-bearing mice. We identified that Siglec-E is expressed on myeloid cells and CD8+ T cells in the tumour microenvironment. However, in spleen, blood and tumour-draining lymph nodes not only CD8+ but also CD4+ T cells expressed Siglec-E. Interestingly, Siglec-E+ and Siglec-E- T cells presented distinct expression of PD-1 across tissues, suggesting different regulation mechanisms for the expression of these immune checkpoints. Our work provides an investigation of the sialoglycans on B16OVA cells and the expression of their receptor Siglec-E across tissues, which is of importance for future therapeutic studies targeting the sialic acids-Siglec axis, especially in combination with anti-PD-1 therapies.
