Glycobiology最新文献

Although the interaction between β-1,3-glucans (BG) and β-1,3-glucan recognition protein (BGRP) derived from insects is well established, the binding interaction and recognition mechanism of BG when complexed with deoxyadenosine (dA) or CpG oligodeoxynucleotides (CpG) remain poorly understood. In this study, we investigated the binding properties of Schizophyllan and Curdlan to BGRP both in their native forms and in BG/DNA complexes. Our findings revealed that BG/dA complexes bind to BGRP via a canonical BG-BGRP binding mechanism, whereas BG/CpG complexes exhibited a recognition mechanism distinct from the BG-BGRP interaction. Structural alterations in BG upon complexation with CpGs appear to induce a unique mode of BGRP recognition. This study reveals a novel mode of BGRP recognition in CpG-containing complexes, offering insights into improved immune detection strategies.

Human milk oligosaccharides (HMOs) are complex sugars. These sugars possess prebiotic, antibiotic and immunomodulatory properties and therefore are important for the health and well-being of newborn babies. The backbones of HMOs are terminated either with Type I LacNAc (Gal-β1,3-GlcNAc) or Type II LacNAc (Gal-β1,4-GlcNAc) that can be further fucosylated and sialylated. To detect all these HMOs including their fucosylated and sialylated versions, we explored enzymatic incorporation of azido-fucose (N3-Fuc) by FUT2 or FUT3 directly, or through a replacement approach where existing fucose and sialic acid are removed with a specific glycosidase and replaced with an N3-Fuc. Specifically, AfcA, an α1,2-linkage specific fucosidase cloned from Aspergillus oryzae, was used to remove existing α1,2-Fuc. The substrate specificities and relative efficiencies of AfcA, FUT2 and FUT3 in terms of the usage of N3-Fuc were demonstrated on standard HMOs. FUT2 was finally selected for labeling and validated on HMOs isolated from human milk samples. Furthermore, using Cy5-labeled antibody glycan G2 as a gel control, the relative gel separation of an N3-Fuc labeled HMO was established, which could aid identification of the oligosaccharide. This strategy by N3-Fuc labeling and glycan electrophoresis expands the ability to profile HMOs and is complementary to traditional methods for HMO study.

Mucin-type O-glycans are abundant protein modifications that regulate cell signalling, adhesion, and immune interactions. In cancer, their biosynthetic pathways are frequently disrupted, leading to the accumulation of truncated structures, such as Tn antigen and sialyl-Tn (STn). These aberrant glycans remodel the glycocalyx, alter receptor clustering, and drive key hallmarks of malignancy, including immune evasion, invasion, and therapy resistance. Over the past decade, increasing evidence has linked short O-glycans to poor prognosis across multiple tumour types, highlighting their potential as diagnostic and prognostic biomarkers. Moreover, their restricted expression in normal tissues positions them as attractive targets for therapeutic intervention, including monoclonal antibodies, antibody-drug conjugates, and CAR-T cell strategies. However, clinical translation remains limited by major analytical challenges. The structural diversity of O-glycans, their low abundance, and the lack of broadly specific enzymes for glycan release hinder comprehensive characterization. Recent advances in glycoproteomics, glycomics, and antibody engineering are beginning to overcome these barriers, enabling site-specific mapping and improved detection of cancer-associated glycoforms. This review summarizes current knowledge on the biosynthetic origins, biological roles, and clinical relevance of truncated O-glycans in cancer, while critically discussing emerging technologies and future directions for their integration into precision oncology.

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a unique type of protein glycosylation that intricately links cellular metabolism to various signaling pathways. This reversible, nutrient-sensitive modification dynamically regulates a wide range of biological processes, including apoptosis, cell proliferation, and differentiation. Recent studies have made substantial progress in elucidating the pivotal roles of O-GlcNAcylation in modulating key oncogenes and signaling cascades. Aberrant O-GlcNAc cycling has been associated with a variety of pathological conditions, including cancer, metabolic disorders, and neurodegenerative diseases, underscoring its critical influence on cell fate decisions. In this review, we will highlight recent advances in understanding how O-GlcNAcylation modulates major cell fate regulating pathways, including nuclear factor kappaB (NF-κB), Notch, G protein-coupled receptor (GPCR) signaling, and transforming growth factor beta (TGF-β). We propose that O-GlcNAcylation integrates extracellular signals with intracellular metabolic states, functioning as an essential "Glyco-Switch" sensor that modulates cell fate decisions in both physiological and pathological contexts.

A major challenge in the glycosciences is the scarcity of sensitive and specific glycan-binding reagents, such as monoclonal antibodies, for detecting and isolating glycans. Here we report the development and characterization of new monoclonal antibodies (mAbs) that bind carbohydrate-based red blood cell (RBC) antigens including the ABO(H) antigens. This approach exploits the immune system of the sea lamprey (Petromyzon marinus), which strongly responds to human glycans to enable the generation of high affinity antibodies. To develop these mAbs, we immunized the lamprey with RBCs and designed a targeted antibody enrichment and screening process using intact RBCs and a custom microarray displaying blood group antigens. Through multiple rounds of enrichment and testing we identified two mAbs; A_25 and A_39. Glycan binding analysis of the mAbs using glycan microarrays, the Luminex platform and western blot analysis revealed their binding to H antigens and terminal N-acetyllactosamine Galβ1-4GlcNAc (LacNAc, a type 2 sequence). Mechanistic insights into antigen specificity were gained through glycan inhibition assays, sequence homology analysis, and nanomolar-range affinity measurements. mAb binding to RBCs was determined using flow cytometry. Both mAbs bound RBCs of all ABO blood groups, whereas strongest binding was observed for blood group O RBCs. Our findings highlight the efficacy of the lamprey system to develop glycan-specific mAbs. These reagents allow investigation of expression of the H antigen and LacNAc-containing glycans in human tissues. In the future, they could also be modified using molecular engineering techniques to generate mAbs specific to other understudied blood group antigens.

GalNAc-Ts are a large family of glycosyltransferases that regulate numerous cellular processes by initiating the post-translational modification mucin-type O-glycosylation. Disruptions in GalNAc-T expression and function are associated with congenital diseases, metabolic disorders, and cancer. The substrates and acceptor sites affected by the inactivation or over-activation of each specific family member are often not known due to acceptor site and substrate redundancies among the isoenzymes that are present within a cell type. However, substantial progress has been made in disentangling the enzyme-substrate conundrum by showing that each isoenzyme follows a unique set of substrate recognition rules. This review summarizes biochemical and structural findings that have advanced our understanding of the distinct substrate specificities of individual GalNAc-Ts.

Glycans regulate a wide array of biological processes, making them central to studies of cell biology. Thus, it is essential to characterize the spatiotemporal dynamics of glycans on cells and tissues, and to elucidate how glycan structures affect protein and cell function. Among the available molecular tools, glycan-binding proteins (GBPs), including naturally occurring lectins, are uniquely suited to provide this information at single-cell resolution. However, the diversity of cell-surface glycans far exceeds the number of readily available GBPs. Moreover, conventional lectins often possess shallow binding pockets that limit their recognition to terminal glycan epitopes, and such recognition often proceeds with low binding affinity. Protein engineering offers a promising strategy to expand GBP specificity, enhance affinity, and introduce novel binding capabilities. Currently, large gaps remain between the available protein design principles and their application to GBP engineering. This has somewhat slowed progress in the development of glycan-targeted tools. In this review, we outline recent efforts that use rational design to inform GBP engineering for specific tasks. We also present methods to select suitable protein scaffolds and the application of directed evolution for optimizing lectin design. This includes our recent efforts to modify glycosyltransferases into GBPs, which potentially offers a predictive strategy to design lectins based on desired properties. Together, the presentation offers a roadmap for developing next-generation glycan binding proteins capable of decoding the complex glycan landscape of cells.

The mammalian brain is unique in its cell types, mainly neurons and glial cells, and the glycoproteins expressed by these cells. Two of the most abundant types of modifications of cell surface glycoproteins are N-glycans linked to Asn residues and O-glycans linked via GalNAc to Ser/Thr residues. Recent studies focused on glycoproteomics, glycomics and glycan localization in the brain reveal major differences in these protein modifications compared to other organs. Deficiencies in glycosylation are associated with the development of multiple brain disorders such as congenital disorders of glycosylation (CDG) that include brain structural abnormalities, epilepsy and seizures to more common disorders including schizophrenia and Alzheimer's disease. Here we summarize recent advances in the growing field of neuro-glycobiology and highlight key points that could be used as primer for future studies.

The interactions between environmental glycans and glycan-binding proteins modulate a host of processes across biological systems. The Siglec/sialic acid axis has gained increasing attention as an immunologic checkpoint due to its involvement with reducing inflammatory processes and promoting tumor growth. Siglec-2, or CD22, has been extensively characterized as a co-receptor for the B cell receptor (BCR) and is critical for the prevention of self-reactive B cell responses through its recognition of α2,6-linked sialic acids. More recently, CD22 has emerged as an important receptor for macrophage biology. Here, we investigate the consequences of genetic ablation of CD22 in murine macrophages (CD22KO). Aged CD22KO mice developed a fatty liver phenotype similar to that seen in aged animals lacking hepatocyte α2,6-sialylation (HcKO). CD22KO bone marrow-derived macrophages (BMDMs) exhibited few differences in canonical markers of M1-like and M2-like polarization, but M2-like CD22KO BMDMs showed a pro-inflammatory shift in transcriptome and a reduction in endocytic and efferocytotic capacity. These data suggest that CD22 in murine M2-like macrophages is strongly associated with a homeostatic transcriptional profile and directly participates in immunologically silent housekeeping functions such as clearance of sialylated-self debris through the Siglec-sialic acid axis.