Glycobiology最新文献

We test the performance of the multiplexed liquid glycan array (LiGA) technology in serological assays. Specifically, we use LiGA to detect ABO blood group antibodies in human serum. This LiGA, which we name ABO-LiGA, contains ABO blood group trisaccharide glycans with an ethylazido aglycone conjugated to groups of ten multi-barcoded M13 particles carrying dibenzocyclooctyne (DBCO) on p8 proteins. ELISA clonal binding assays to anti-A/B antibodies confirmed the functional performance of ABO-clones and aligned with next-generation sequencing (NGS) of the mixed clones. Multiple DNA-barcoded technical replicates in LiGA allow for quantification of reproducibility and robustness as determined by the Z'-score using NGS. We then tested ABO-LiGA for specific detection of IgG and IgM anti-A and anti-B IgG and IgM antibodies in human serum samples. Comparison of antibody binding responses in sera from 31 healthy donors to ABO-LiGA with an ABO-Luminex-based method revealed consistent responses to LiGA-ABO but also minor deficiencies of ABO-LiGA such as low robustness of the current assay format and a limited ability to detect low intensity antibody responses. Some results point to undesired interactions of serum antibodies with small-footprint glycans conjugated to phage via the bulky DBCO moiety. This report illuminates the path for future development of LiGA-based serological assays and suggests the need to develop alternative methods for conjugating glycans to phage to avoid liabilities of the hydrophobic DBCO moiety.

Bacterial vaginosis (BV), a dysbiosis of the vaginal microbiome, affects approximately 30% of women worldwide (up to 50% in some regions) and is associated with several adverse health outcomes including preterm birth and increased incidence of sexually transmitted infections (STIs). BV-associated bacteria such as Gardnerella vaginalis and Prevotella timonensis damage the vaginal mucosa through the activity of sialidase enzymes that remodel the epithelial glycocalyx and degrade mucin glycoproteins. This damage may contribute to adverse health outcomes. However, whether BV-associated glycolytic enzymes also damage sperm has not yet been determined. Here, we show that sialidase-mediated glycocalyx remodeling of human sperm increases sperm susceptibility to damage and adversely affects their function in vitro. Specifically, we report that sperm motility was not adversely affected by sialidase treatment, but desialylated human sperm demonstrate increased susceptibility to agglutination and complement-mediated cytotoxicity as well as impaired transit through cervical mucus. Our results demonstrate mechanisms by which BV-associated sialidases affect sperm survival and function and potentially contribute to adverse reproductive outcomes such as infertility.

Layilin, an understudied C-type lectin receptor for hyaluronan, was initially hypothesized to regulate cell motility due to its binding partner, talin. Subsequent studies identified layilin as a receptor for hyaluronan with roles in regulating cell motility through interactions with key regulatory molecules upstream of cytoskeletal rearrangement: radixin, merlin, focal adhesion kinase (FAK), F-actin, and small GTPases such as RAC1, RAP1, and RhoA. Layilin is also associated with cell-cell interactions, co-localizing with integrins in both T-cells and platelets contributing to epithelial cell junction integrity. Recent studies have found that layilin also plays a role in inflammation, dependent on tissue and disease. In the context of cancer, multiple cancer cell types displaying increased layilin expression contributes to enhanced metastasis. Exhausted CD8+ T cells residing in the tumors exhibit high expression of layilin, with the receptor contributing to increased tissue anchoring and co-expressing with immune checkpoint resistance markers. In other contexts, such as inflammatory bowel disease and atherosclerosis, reduction of layilin results in worsened disease and inflammation. Transcriptomic and epigenetic studies have explored layilin as a prognostic marker, as layilin expression is elevated in multiple cancers, deep vein thrombosis, diabetes, and Alzheimer's. However, the mechanistic role of layilin in most of these studies remains unexplored. This review outlines current insights into Layilin as a molecular hub that links hyaluronan signaling with integrin activity and cytoskeletal dynamics, highlighting its roles in homeostasis, pathogenesis, disease prognosis, and therapeutic intervention across diverse conditions.

Bacterial infections represent a substantial global health challenge, impacting both human and veterinary health. The ongoing evolution of antibiotic-resistant pathogens, coupled with limited new antibiotic discoveries, urges the need for alternative strategies to treat and prevent these infections. Passive immunization with monoclonal antibodies (mAbs) is gaining interest as a promising alternative. Here, we report an experimental pipeline for generating human mAbs from healthy donor B cells using synthetic mimics of complex bacterial glycans. We identified functional mAbs recognizing discrete and unique epitopes on the surface glycans of two bacterial priority pathogens; Staphylococcus aureus and Streptococcus pyogenes. The use of chemically-defined synthetic glycans was critical for the discovery and systematic characterization of mAbs. From a heterogeneous mix of B cell specificities, antibody sequences were identified, leading to the production of mAbs with distinct reactivities against immunodominant but also to less common or even masked epitopes. The pipeline can be adapted to different glycan targets, donor material or specific antibody isotypes. This work thereby paves the way for the discovery of glycan-specific mAbs with clinical relevance to treat, prevent or diagnose infections with S. aureus, S. pyogenes or other bacterial pathogens.

Glycomics and glycoproteomics represent the systematic exploration of glycan structures and glycoprotein compositions within biological systems, aiming to elucidate their roles in physiological and pathological processes, including cancer, inflammation and infectious diseases. To support this investigation, glycomics and glycoproteomics utilize a diverse array of methodologies from molecular biology, biochemistry, analytical chemistry and bioinformatics. In this study, we investigated the semantic representation experimental workflows in glycomics and glycoproteomics publications through graph-based annotation using combination of existing domain-relevant ontologies. Rather than adhering to evolving metadata standards, this investigation explored a broad spectrum of biomedical and analytical ontologies to identify optimal annotations for the generative (e.g. sample preparation and derivatization) and transformative (e.g. separation and detection) phases of the workflow. The results show that integrating several ontologies yields more precise annotations than relying on a single one. However, several challenges arose, particularly where methodological reporting lacked critical metadata, such as derivatization conditions or glycan release protocols. Furthermore, the annotations imply that methodologies in the glycomic and glycoproteomic fields are more complex, on average, than those in other scientific fields. The results suggests that, while some specific concepts are missing in the ontologies, a limited number of ontologies adequately encompass the majority of aspects related to glycomics and glycoproteomics experiments. These can serve as a foundation for community-wide metadata standards and direct future efforts to refine and expand the ontologies for glycoscience research.

Dectin-1, a C-type lectin receptor that recognizes β-glucans, plays a vital role in antifungal immunity and is involved in inflammatory diseases and cancer, making it a promising therapeutic target for antagonists. However, current evaluations of these antagonists often depend on binding inhibition assays, which may not accurately reflect physiological functional suppression. This study addresses the need for a rapid, functional, cell-based assay for human Dectin-1 (hDectin-1) antagonists. We describe the development and validation of such an assay using THP-1 cells stably expressing hDectin-1 (dTHP-1 cells), which produce TNF-α upon Dectin-1 activation by depleted Zymosan (dZymosan). We established optimal assay conditions as 10 μg/mL dZymosan stimulation for 4 h. Under these conditions, laminarin, a soluble β-glucan, inhibited dZymosan-induced TNF-α production in a dose-dependent manner. This inhibition was specific, as Dextran T40, a non-Dectin-1-binding polysaccharide, had no inhibitory effect on dZymosan-induced responses. This novel, concise (4-hour) assay system directly measures the key physiological outcomes of Dectin-1 signaling, offering a significant improvement over binding-based assays. This provides a valuable platform for screening and characterizing hDectin-1 antagonists, facilitating the development of new therapeutics for Dectin-1-related pathologies.

Core fucosyltransferase is crucial for core fucosylation modification, and its expression is upregulated in various types of cancers. This upregualtion is involved in tumorigenesis and development as well as closely related to cancer diagnosis and prognosis. In recent years, core fucosylation pathway has been extensively studied, which is imperative for the development of antibodies targeting core fucosylation. This study focuses on the regulatory mechanism of core fucosylation and summarizes the research results of related inhibitors, including methods to inhibit substrate and develop fucosyltransferase 8 inhibitors. This study also comprehensively reviews preparation techniques for deglycosylated antibodies, including gene-editing technology, exogenous addition of soluble RNA, bicistronic gene design, and synthesis of glycosylation inhibitors. Moreover, the current challenges in this field are also investigated in this study, and future research directions are proposed to provide a comprehensive reference for the research on core fucosylation in cancer therapy and related antibody engineering fields.