Glycobiology最新文献

Toxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.

Glycans are complex carbohydrates that exhibit extraordinary structural complexity and stereochemical diversity while playing essential roles in many biological processes, including immune regulation, pathogen recognition, and cell communication. In humans, more than half of all proteins are glycosylated, particularly those in secretory and membrane-associated pathways, highlighting the importance of glycans in health and disease. The recent release of the AlphaFold 3 source code enables customizable modeling not only of proteins but also glycan-containing biomolecular complexes. We assessed the capacity of AlphaFold 3 to model glycans using several input formats and identified a hybrid syntax employing Chemical Component Dictionary (CCD)-based molecular building blocks linked by "bondedAtomPairs" (BAP) as most effective in generating stereochemically valid glycan models. This workflow was used to create a library of AlphaFold 3 input templates and corresponding structural models for various glycan classes. We further explored capabilities, limitations, and remediation strategies for modeling problematic structures. Glycan interactions were also modeled with glycosylation enzymes and lectins with benchmarking and validation against known crystal structures. This protocol-driven approach is valuable for generating stereochemically valid, static models of glycan-protein interactions to support hypothesis development and subsequent structural and functional validation. However, caution should be observed in overinterpretation of the static models since glycans are known to exhibit considerable conformational dynamics that can be further captured by equilibrium sampling using molecular dynamics-based approaches. By sharing benchmarked examples using the BAP syntax we aim to support broader evaluation of AlphaFold 3 in studying glycan-related mechanisms in biosynthesis, signaling, infection, and disease.

Changes in glycosylation can serve as markers for rare genetic disorders, including lysosomal storage diseases (LSDs). Nephropathic Cystinosis (NC), caused by mutations in the CTNS gene, is characterised by cystine accumulation in lysosomes due to dysfunctional cystinosin, a heavily N-glycosylated lysosomal transporter. We analysed total serum and IgG N-glycosylation using hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) to explore the diagnostic biomarker capabilities and their pathophysiological relevance in NC. In this double-blind study (n = 12), we examined N-glycosylation of total serum and serum IgG from Irish participants with and without NC. Dimensionality reduction methods were used applying their glycan data to predict NC status, yet only modest predictive power was observed (66.6% for serum and 50% for IgG N-glycosylation). However, upon unblinding the data, we identified significant differences in specific serum N-glycosylation in NC, particularly in sialylation. These findings provide the first evidence that serum N-glycosylation is altered in NC. These changes may indicate disease-associated systemic alteration including dysregulation in N-glycosylation pathway. It provides justification for the need for a larger validation study and invites further exploration of its role in NC pathophysiology. We provide key recommendations for age stratification for studying serum, plasma and IgG N-glycans in juvenile cohorts as they display unique profiles compared to adult populations, an important consideration for all juvenile studies, even beyond the scope of rare diseases.

The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked β-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.

The H11 antigens, located on the intestinal microvilli of Haemonchus contortus, comprise a group of homologous aminopeptidases essential for the parasite's digestion of blood meals. Native H11 proteins are promising vaccine antigens, capable of eliciting robust protective immunity against H. contortus in sheep and goats. However, recombinant forms of H11, produced either in conventional expression systems or in transgenic Caenorhabditis elegans, failed to replicate the protective efficacy of the native form, most likely due to two critical factors: improper glycosylation and protein misfolding. To address these limitations, we developed a novel strategy to produce recombinant Haemonchus antigens in glycoengineered insect cells. By introducing three C. elegans genes that alter the native N-glycosylation pathways of Hi5 insect cells we successfully expressed soluble H11 and GA1 antigens featuring nematode-specific glycan epitopes, including tri-fucosylated structures and the Galβ1,4Fuc motif. The glycoengineered H11 proteins retained aminopeptidase activity and stimulated cytokine secretion from ovine peripheral blood mononuclear cells in vitro. These findings establish a platform for producing bioactive vaccine antigens against the parasitic nematode H. contortus.

Excitatory and inhibitory synapses are the two major fundamental units of neuronal communication in the brain. The imbalance between excitatory and inhibitory synapses (E/I imbalance) is a leading mechanism underlying mental illness. Heparan sulfate (HS), a complex polysaccharide frequently implicated in mental disorders, is an emergent player in synaptic function. Yet, it remains unclear whether and how HS plays a preferential role in excitatory versus inhibitory synapses. This question is further complicated by the structural complexity of HS and the combined effects of both HS glycans and their attached proteoglycans. To address this challenge, we developed a platform that combines synthetic chemistry and synaptic biology to dissect the role of pure HS glycans in synapse development. As proof of principle, we assessed the effects of a synthetic dodecasaccharide (12-mer-19) and its non-sulfated counterpart (12-mer-NAc) on excitatory and inhibitory synapses in primary rat hippocampal neuron cultures. Unexpectedly, we found that 12-mer-19 selectively impaired the morphology and function of excitatory but not inhibitory synapses. Mechanistically, 12-mer-19 interferes with the interaction between neurexin1 and its partners at excitatory synapses, but has little effect on neurexin1's partner at inhibitory synapses. Moreover, 12-mer-NAc didn't have such effects, highlighting the importance of sulfated groups. Our results suggest that extracellular complex glycans may have a selective yet underappreciated role in excitatory synapses, perhaps contributing to the E/I imbalance. Moreover, current studies lay a foundation for future work to dissect the contribution of specific heparan sulfate structures to synaptic morphology and function.

Male reproduction is a complex process governed by sophisticated cellular and molecular pathways including sperm production, maturation, and delivery. This review underscores the indispensable role of gangliosides-sialic acid-bearing glycosphingolipids prevalent in the male reproductive system-as key modulators of sperm functionality. Gangliosides, which consist of a ceramide core linked to oligosaccharide chains, are predominantly found on cell plasma membranes, where they play crucial roles in cell signaling, adhesion, recognition, and membrane structure. Extensive research has revealed gangliosides' dynamic contributions to various facets of sperm physiology, such as maturation, capacitation, the acrosome reaction, and ultimately, fertilization. Variability in ganglioside composition and localization during different sperm development stages and within specific areas of the male reproductive tract underscores their importance in sperm functionality and reproductive outcome. Furthermore, disruptions in ganglioside synthesis, transport and distribution on the membrane or surrounding molecules have been associated with male infertility and reproductive dysfunctions, positioning them as potential biomarkers for these conditions. The findings presented in this review not only advance our understanding of the biochemical landscape of male fertility but also propose gangliosides as potential targets for therapeutic intervention, offering a promising avenue for addressing male reproductive disorders. The exploration of gangliosides in the context of male reproduction not only enhances our understanding of male fertility but also paves the way for novel diagnostic and therapeutic strategies in reproductive health. These insights emphasize the urgency and significance of further investigative efforts into ganglioside functions to potentially revolutionize the diagnosis and treatment of male reproductive abnormalities.