Glycobiology最新文献

The sugar-binding receptor mincle stimulates macrophages when it encounters surface glycans on pathogens, such as trehalose dimycolate glycolipid in the outer membrane of mycobacteria. Binding of oligosaccharide ligands to the extracellular C-type carbohydrate-recognition domain (CRD) in mincle initiates intracellular signaling through the common Fc receptor γ (FcRγ) adapter molecule associated with mincle. One potential mechanism for initiation of signaling involves clustering of receptors, so it is important to understand the oligomeric state of mincle. Affinity purification of mincle from transfected mammalian cells has been used to show that mincle exists as a pre-formed, disulfide-linked dimer. Deletion of cysteine residues and chemical crosslinking further demonstrate that the dimers of mincle are stabilized by a disulfide bond between cysteine residues in the neck sequence that links the CRD to the membrane. In contrast, cysteine residues in the transmembrane region of mincle are not required for dimer formation or association with FcRγ. A protocol has been developed for efficient production of a disulfide-linked extracellular domain fragment of mincle in a bacterial expression system by appending synthetic dimerization domains to guide dimer formation in the absence of the membrane anchor.

Nictaba is a (GlcNAc)n-binding, stress-inducible lectin from Nicotiana tabacum that serves as a representative for the Nictaba-related lectins, a group of proteins that play pivotal roles in plant defense mechanisms and stress response pathways. Despite extensive research into biological activities and physiological role(s) of the lectin, the three-dimensional structure of Nictaba remained largely unknown. Here, we report crystal structures for Nictaba in the apo form and bound to chitotriose. The structures reveal that the Nictaba protomer has a jelly-roll fold, similar to the cucumber lectin Cus17, but exhibit a unique and previously unseen mode of dimerization. The chitotriose binding mode, similar to Cus17, centers around the central GlcNAc residue, providing insights into the determinants of specificity of Nictaba towards carbohydrate structures. By integrating these structural insights with inputs from glycan arrays, molecular docking, and molecular dynamics simulations, we propose that Nictaba employs a single carbohydrate-recognition domain within each of the two subunits in the dimer to display pronounced specificity towards GlcNAc-containing carbohydrates. Furthermore, we identified amino acid residues involved in the extended binding site capable of accommodating structurally diverse high-mannose and complex N-glycans. Glycan array and in silico analyses revealed interactions centered around the conserved Man3GlcNAc2 core, explaining the broad recognition of N-glycan structures. Collectively, the structural and biochemical insights presented here fill a void into the atlas of lectin structure-function relationships and pave the way for future developments in plant stress biology and lectin-based applications.

Escherichia coli serotype O117 (ECO117) are pathogenic bacteria that produce Shiga toxin. Repeating units of the O antigen of ECO117 have the pentasaccharide structure [4-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-]n. The related non-pathogenic serotype (ECO107) contains a GlcNAc residue instead of Glc in the repeating unit, and the biosynthetic enzymes involved are almost identical. We assembled these repeating units based on GalNAcα-diphosphate-phenylundecyl (GalNAcα-PP-PhU), an analog of the natural intermediate GalNAc-diphosphate-undecaprenyl. We previously characterized α1,4-Glc-transferase WclY from ECO117 that transfers the Glc residue to Galβ1-3GalNAcα-PP-PhU and showed that Arg194Cys mutants of WclY are active α1,4-GlcNAc-transferases. In this work, the reaction products of WclY were used as acceptor substrates for the final enzymes in pathway, L-Rha-transferase WclX, and GalNAc-transferase WclW, demonstrating a complete synthesis of the ECO117 and O107 repeating units. WclX transfers L-Rha with high specificity for the WclY enzyme product as the acceptor and for TDP-L-Rha as the donor substrate. A number of highly conserved sequence motifs were identified (DDGSxD, DxDD, and YR). Mutational analysis revealed several Asp residues are essential for the catalysis of L-Rha transfer, while mutations of Asp44 and Arg212 substantially reduced the activity of WclX. WclW is a GT2 enzyme specific for UDP-GalNAc but with broad specificity for the acceptor substrate. Using L-Rhaα-p-nitrophenyl as an acceptor for WclW, the reaction product was analyzed by NMR demonstrating that GalNAc was transferred in a β1-3 linkage to L-Rha. The in vitro synthesis of the repeating units allows the production of vaccine candidates and identifies potential targets for inhibition of O antigen biosynthesis.

It remains a mystery why nature evolved the unique structural characteristics of GPI-anchored proteins (GPI-APs) and continues to sustain the complex, energy-intensive process of synthesizing these proteins. GPI-APs, despite their small size, rely on the coordinated activity of nearly 30 genes for their synthesis and remodeling, raising important evolutionary questions. The biological advantages of GPI-APs lie in their ability to rapidly redistribute across the cell membrane, localize within lipid rafts, utilize unique intracellular trafficking pathways, and function as both membrane-bound and soluble proteins. These properties allow GPI-APs to participate in diverse cellular processes such as synaptic plasticity, immune regulation, and signal transduction, highlighting their indispensable roles. Additionally, the shedding capability of GPI-APs extends their functional reach, adding further versatility to their biological roles. This review not only summarizes these key insights but also explores the broader implications of GPI-APs in cell signaling and disease. By understanding the evolutionary necessity of GPI-APs, we can better appreciate their complexity and potential as therapeutic targets.

The sugar-binding receptors dectin-2 and blood dendritic cell antigen 2 (BDCA-2) bind oligosaccharide ligands through extracellular carbohydrate-recognition domains (CRDs) and initiate intracellular signaling through Fc receptor γ adapters (FcRγ). Dectin-2 stimulates macrophages in response to pathogen binding while BDCA-2 modulates cytokine production in plasmacytoid dendritic cells. The oligomeric states of these receptors and the orientations of their CRDs have been investigated by analysis of a naturally occurring disulfide-bonded variant of BDCA-2 and by replacement of transmembrane domains with N-terminal dimerization domains to create extracellular domain dimers of both dectin-2 and BDCA-2. Analysis of these constructs, as well as previously described crystal structures of the CRDs from these proteins and a novel structure of an extended version of the extracellular domain of dectin-2, showed that there is only limited interaction of the CRDs in the dimers, but interactions can be stabilized by the presence of the neck region. The resulting orientation of sugar-binding sites in the dimers would favor crosslinking of multiple dimers by oligosaccharide ligands, causing clustering of FcRγ to initiate signaling.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P < 0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

Immunopeptides are cell surface-located protein fragments that aid our immune system to recognise and respond to pathogenic insult and malignant transformation. In this two-part communication, we firstly summarise and reflect on our recent discovery documenting that MHC-II-bound immunopeptides from immortalised cell lines prevalently carry N-glycans that differ from the cellular glycoproteome (Goodson, Front Immunol, 2023). These findings are important as immunopeptide glycosylation remains poorly understood in immunosurveillance. The study also opened up new technical and biological questions that we address in the second part of this communication. Our study highlighted that the performance of the search engines used to detect glycosylated immunopeptides from LC-MS/MS data remains untested and, importantly, that little biochemical in vivo evidence is available to document the nature of glycopeptide antigens in tumour tissues. To this end, we compared the N-glycosylated MHC-II-bound immunopeptides that were reported from tumour tissues of 14 meningioma patients in the MSFragger-HLA-Glyco database (Bedran, Nat Commun, 2023) to those we identified with the commercial Byonic software. Encouragingly, the search engines produced similar outputs supporting that N-glycosylated MHC-II-bound immunopeptides are prevalent in meningioma tumour tissues. Consistent also with in vitro findings, the tissue-derived MHC-II-bound immunopeptides were found to predominantly carry hyper-processed (paucimannosidic- and chitobiose core-type) and hypo-processed (oligomannosidic-type) N-glycans that varied in prevalence and distribution between patients. Taken together, evidence is emerging suggesting that α-mannosidic glycoepitopes abundantly decorate MHC-II-bound immunopeptides presented in both immortalised cells and tumour tissues warranting further research into their functional roles in immunosurveillance.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1-/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

The Human Glycome Atlas (HGA) Project was launched in April 2023, spearheaded by three Japanese institutes: the Tokai National Higher Education and Research System, the National Institutes of Natural Sciences, and Soka University. This was the first time that a field in the life sciences was adopted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for a Large-scale Academic Frontiers Promotion Project. This project aims to construct a knowledgebase of human glycans and glycoproteins as a standard for the human glycome. A high-throughput pipeline for comprehensively analyzing 20,000 blood samples in its first five years is planned, at which time an access-controlled version of a human glycomics knowledgebase, called TOHSA, will be released. By the end of the final tenth year, TOHSA will provide a central resource linking human glycan data with other omics data including disease-related information.