O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that widely present in eukaryotic cytoplasmic proteins or mitochondria. It affects protein properties, cell functions, disease states and so on, hence it is import to identify the roles that O-GlcNAcylations play on target proteins in living cells. The new technologies for manipulating O-GlcNAcylations on target proteins will greatly accelerate the understanding of O-GlcNAc’s functions. This article briefly introduces some recent research progress of chemical biology techniques for targeted protein O-GlcNAcylation, and also analyzes the advantages and limitations of these strategies, and their future development prospects. These technologies build a powerful chemical biology toolbox, which may contribute to the diagnoses and treatments related to O-GlcNAcylation.

Covalent attachment of glycans to the side chains of protein amino acids can form glycosylation. Glycosylation, in many cases, significantly increases the diversity of protein structure, properties, and functions. Specific glycosylation patterns can confer specific effects on proteins and enzymes, while aberrant glycosylation patterns may lead to diseases. Therefore, a comprehensive understanding of the role of protein glycosylation is of great significance, both for basic and applied research. However, progress in this area has been exceptionally slow due to the difficulty in obtaining suitable samples for study. In recent years, researchers have gradually begun to explore the use of a library-based strategy to change this situation. This strategy involves the synthetic preparation and characterization of a series of homogeneously glycosylated protein isoforms (glycoforms) with high purity and systematic variations in structures. Through the following comparative analysis, the effects and functioning mechanisms of protein glycosylation can be relatively quickly and accurately obtained. This, in turn, enhances the application of glycosylation in improving the performance of proteins and enzymes. This review aims to summarize, outline, and briefly discuss the progress in the O-glycosylation research direction. It presents research methods and achievements of a few existing examples in chronological order, with the aim of helping researchers gain a clearer understanding of the current development and shortcomings of this strategy. This review is expected to assist researchers in better utilizing this strategy for protein glycosylation research and applications, ultimately improving the depth and breadth of this research.

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a widespread post-translational modification of intracellular proteins. Unlike common types of protein glycosylation, O-GlcNAc transferase (OGT) adds a single GlcNAc unit to serine or threonine residue of proteins. Since its discovery, a large number of studies have shown that O-GlcNAcylation is widely involved in many fundamental physiological processes such as cell growth and development, gene transcription, immune response and stress response. In the immune system, O-GlcNAcylation regulates the activation, differentiation and function of immune cells through various ways. The differentiation and phenotypic maintenance of macrophages are dependent on O-GlcNAcylation, the fluctuation of glucose metabolism levels or the loss of OGT will lead to the transformation of macrophage polarization. In addition, O-GlcNAcylation regulates cytokines transcription by altering the activity of transcription factors such as NF-κB to maintain macrophage inflammation, and also affects MAVS (mitochondrial antiviral signaling) protein ubiquitination in response to pathogen infection. In other innate immune cells, the reduction of O-GlcNAcylation will affect cellular immune function to varying degrees. O-GlcNAc regulates transcription factors such as NF-κB, NFAT (nuclear factor of activated T cells) and c-Myc in T cells and B cells, affects the expression of cytokines and metabolism-related genes, and meet cell activation and proliferation by a higher level of glucose intake. The abnormal changes of O-GlcNAcylation in the immune system are closely related to the occurrence and development of chronic inflammation, tumor and other related diseases, or become a means of tumor immune escape. An in-depth understanding of the role of O-GlcNAc modification in the immune system will help to reveal the molecular mechanism of immune regulation and provide a theoretical basis for the development of novel immunotherapy strategies.

糖作为生命体的重要组成成份,承载着很多生物学功能:保护细胞、细胞的结构成份、能量来源与代谢组分、细胞与细胞的识别以及细胞内的信号转导。最常见的人类ABO血型的分子基础就是由血细胞表面的糖链结构决定的,流感病毒侵入宿主细胞则是由宿主细胞表面的唾液酸糖作为受体。
虽然人们很早就认识到了糖的重要性,但其固有的复杂性造成了很多研究上的技术难题:其一,化学结构上,糖链的链接方式多种多样;其二,糖链的合成没有模板,而是如同“分子乐高”一样由糖基转移酶装配合成。这样的糖类异质性虽然可以承载更多的信息,但是也为研究它的生物学功能造成了不小的挑战。
所以糖生物学从诞生的那一刻起,就注定了是一门多学科多手段多角度的交叉学科。本专刊涵盖多学科(生命、化学以及药学)对于糖生物学功能的综述以及技术上的最新简报,力图报道糖科学研究的新技术新方法,阐释糖在不同生理和病理中的功能,并讨论糖类药物的开发。
本专刊一共收录了9篇糖生物学相关的综述以及1篇技术简报。
(1) “工欲善其事,必先利其器”,我们首先综述了研究糖生物学的技术手段:《基于分子库策略的蛋白质O-糖基化研究》通过化学合成的方法来研究不同Ser/Thr位点进行O-糖基化对蛋白质功能的影响和调控;《酶介导的邻近细胞标记方法探究细胞间相互作用》阐释了化学生物学的研究策略;《基因编辑技术及其在糖生物学研究中的应用》则将糖与基因编辑相结合,对未来进行了展望。
(2) 聚焦N-乙酰葡萄糖胺 (O-linked β- N -acetylglucosamine, O-GlcNAc)。O-GlcNAc为发生在细胞内参与信号转导的单糖修饰。综述《靶向编辑O-GlcNAc糖基化修饰的化学生物学技术》从化学的角度阐释了最新的化学生物学工具;技术简报《CpOGA^D298N与核心链霉亲和素(Stv13)融合表达用于检测蛋白O-GlcNAc修饰》则从去N-乙酰葡萄糖胺修饰的角度研究了新的检测方法;《O-连接-N-乙酰葡糖胺糖基化蛋白质的富集方法》介绍了近年来 O-GlcNAc 糖基化修饰与疾病之间的关系以及相关修饰位点的富集方法;《O-GlcNAcylation在免疫系统中的作用》则侧重其在生物学中的免疫功能。
(3) 阐释糖类分子在不同病理中的功能:《免疫分子的糖基化修饰与重要感染性疾病》集中于免疫系统;《溶酶体半乳糖苷酯酶作用机制及疾病》侧重糖在溶酶体这一独特的细胞器中的功能。以此为基础,《糖药物在疾病治疗中的应用》综述了针对糖类的药学研究。
我们相信通过多学科对于糖生物学的交叉探索与创新实践,糖生物学必将迎来崛起的新时代。

The elucidation of gene functions is a fundamental task in modern biological research, the precisive, efficient, and targeted editing of genes is an indispensable tool for their functional dissection. Over the past 30 years, gene editing has experienced the transformation from homologous recombination repair-based technology to programable nuclease-based method, such as zinc-finger nucleases, transcription activator-like effector nucleases, and CRISPR-associated nucleases. The development of these technologies has greatly advanced the investigation of gene functions and led to the emergence of disruptive technologies for disease treatment. In this manuscript, we first introduced the classification, composition, and working principle of CRISPR-Cas for the immune defense in bacteria. We subsequently focus on the state-of-the-art gene editing tools based on the CRISPR-Cas system including the CRISPRi/CRISPRa, base editors, precursor editors, and the RNA-targeted RCas editing system. We then review the strategies for the delivery of gene editors to the desired target cells/organs, we mainly discuss the pros and cons of adeno-associated viruses, lipid nanoparticles, and extracellular vesicles. Finally, we review the applications of gene editing technologies in glycobiology research, including the function, biosynthesis, and underlying mechanism for the carbohydrates, glycoproteomic analysis, the construction and application of cellular glycan array, and the protein glycoengineering. In conclusion, the development of the precise gene editing technology has significantly promoted the research on the biosynthesis, structure, and function of carbohydrates, which has also advanced the translational aspect of glycoscience research.

Carbohydrates play a crucial role in various life processes such as cell recognition, bacterial infection, signal transduction, and immune response. Due to their associated biological effects, the naturally existing carbohydrates and their derivatives have been extensively studied. Carbohydrates are also important lead for drug development, carbohydrate-based drugs exhibit excellent therapeutic efficacy in the treatment of various diseases such as infection, cancer, and cardiovascular disease. Ribose and deoxyribose are the primary scaffolds for nucleoside drugs, which can inhibit viral replication and serve as treatments for viral infections. Carbohydrates-containing macrolide and aminoglycoside antibiotics target the 50S subunit and 30S subunit of bacterial ribosomes respectively, to hinder protein synthesis and eliminate bacteria. Heparin, a highly sulfated glycosaminoglycan, acts as an anticoagulant by binding to antithrombin III and inactivating thrombin. Pseudo-saccharide can bind to glycosidase to prevent oligosaccharide hydrolysis, thereby controlling blood sugar levels. Additionally, sugar vaccines are crucial in cancer treatment, highlighting the broad applications of sugar drugs across various diseases. Furthermore, the rapid development of glycochemistry has deepened scientists’ understanding of carbohydrates, and medicinal chemists increasingly apply this knowledge to the design of new drugs. This review provides a brief overview of the application of carbohydrate-containing drugs in various disease.

Cell-cell interactions (CCIs) can occur through the formation of intercellular synapses mediated by cell surface proteins, glycans, lipids, to maintain body homeostasis and regulate physiological functions.These CCIs are complex, involving the participation of many different cell surface and intracellular molecules. Therefore it is key to accurately identify, characterize and quantify cell-cell interactions. In recent years, the technical means of researching CCIs have been continuously introduced, among which proximity labeling is a promising chemical biology method for studying cell-cell interactions.Currently, there are mainly two types of labeling strategies. One is to rely on the direct binding between enzymes expressed on the surface of “bait” cells by genetic engineering and receptor substrates on adjacent cells to achieve intercellular proximity labeling. The other is to use enzymes or organocatalysts (such as photocatalysts) that are recombinantly expressed by genetic engineering or coupled to the surface of “bait” cells by chemical (chemoenzymatic) methods, and following appropriate stimulation or activation, targeted delivery of labeling molecules are carried out. Between them, the enzyme-mediated proximity cell labeling methods have promising application value in the detection and characterization of CCIs.This review defines methods involving enzymes during the labeling process as enzyme-mediatedproximity cell labeling methods. A remarkable advantage of this approach is the small labeling radius that can be achieved due to direct physical contacts between the enzymes and receptor substrates or enzyme-catalyzed generation of highly reactive labeling molecules.We summarize the principles, advantages and disadvantages,and existing applications of the enzyme-mediated proximity cell labeling methods developed in recent years.

Protein glycosylation, one of the most common post translational modifications (PTMs) of proteins, is widely present in living organisms. In eukaryotic cells, glycosylation modifications have a significant impact on protein folding, conformation, distribution, stability and activity. The glycosylation of glycoproteins is crucial for maintaining the order of interactions between all differentiated cells in multicellular organisms. The dysfunction of protein glycosylation may lead to the occurrence of diseases and development of infectious diseases. So far, changes in protein N/O-glycan have been identified as biomarkers for the development of tumors and certain infectious diseases. Therefore, this article reviews the glycosylation of major immune molecules such as B-cell receptor (BCR), T-cell receptor (TCR), cytokines (CK), complement and immunoglobulin (Ig), as well as the relationship between these glycosylation and infectious diseases. The aim is to understand the association between glycosylation of immune molecules and infectious diseases, and to provide new ideas and strategies for the treatment of infectious diseases.

Lysosomal storage diseases (LSD) are a category of genetic metabolic disorders, originating from genetic mutations in lysosomal acid hydrolases, leading to enzymatic deficiencies. Consequently, this triggers an abnormal accumulation of biological macromolecules within lysosomes, subsequently causes significant damage to cellular, tissue, and organ functions. Mutations or deficiencies in β-galactocerebrosidase (GALC) result in the accumulation of psychosine, causing progressive demyelination and triggering Krabbe Disease, a form of neurodegenerative lysosomal storage disease. The specific mechanisms involved in disease regulation are not yet fully elucidated. Increasing reports of pathogenic mutations in the GALC gene, coupled with analyses of GALC protein structure, have gradually enhanced the understanding of how GALC mutations contribute to Krabbe Disease. This knowledge offers robust evidence for the development of potential therapeutic drugs. Furthermore, GALC plays a dual role in various tumor processes, acting as a tumor suppressor in some cancers while acting as a carcinogen in others. However, a comprehensive analysis of GALC’s impact on cancer requires further in-depth research, offering insight into GALC as a potential target for tumor promotion or suppression. GALC is also associated with various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. Due to the complexity of GALC’s mechanisms, current treatments for Krabbe Disease caused by GALC deficiency primarily involve single-modality and multi-modality therapies. Nevertheless, developing truly effective treatments necessitates deeper research into the pathogenic mechanisms arising from GALC gene defects. This review summarizes the structural and functional characteristics of GALC, and discusses its roles in the development of the nervous system and tumorigenesis, as well as the latest advances in related research. The aim is to lay the theoretical foundation and provide references for exploring GALC’s regulatory mechanisms and developing innovative drugs for treating associated diseases in the future.

O-linked-N-acetylglucosamine (O-GlcNAc) glycosylation is an abundant and unique post- translational modification by adding N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins, which is closely related to various diseases such as cancer, neurodegeneration and diabetes. Identification of the O-GlcNAc modification sites is a prerequisite for exploring their potential regulatory mechanisms in related diseases, as well as a key to clinical diagnosis and targeted intervention. In this paper, we summarize the progresses of diseases associated with aberrant O-GlcNAc modification in recent years, as well as the methods for identifying the relevant modification sites, including antibodies, lectins, chemoenzymatic assays, hydrophilic interaction liquid chromatography, and unnatural carbohydrate metabolism labeling, etc. Furthermore, the strategies of "bump and hole theory" and "spatial activation" have been attracted much attentions recently to targeted label proteins in expected tissues. In addition, multifunctional enzymes theory of "one stone, many birds" and the methods of simultaneous analysis of multiple glycan structures in “one pot” will greatly promote the development of glycoproteomics identification. In conclusion, the development of more effective and specific enrichment approaches for O-GlcNAcylated proteins will be of great significance to elucidate the regulatory mechanism of aberrant glycosylation modifications in related diseases.

Oxygen-linked β-N-acetylglucosamine (O-GlcNAc) modification is a dynamic, reversible post-translational modification that occurs on the hydroxyl group of protein serine and threonine residues and plays important roles in many key cellular processes. Antibodies are commonly used in the detection of protein O-GlcNAcylation, but their specificity against O-GlcNAcylation or the molecular weight range of detected proteins remains to be improved. The Clostridium perfringens OGA mutant (CpOGA^D298N) has been applied in the detection of O-GlcNAcylation in a far-Western blot (Far-WB), due to its advantage in the binding with O-GlcNAc. In this study, CpOGA^D298N was fused with a core streptavidin Stv13. Based on the specific binding property between biotin and streptavidin, we established a fast O-GlcNAc Far-WB assay method, which is verified using short OGT (sOGT), de-O-GlcNAc sOGT, cell lysates and HNF1A proteins. Taken together, our work provides a specific and time-saving Far-WB method (5-7 hours), which can be effectively used in the detection of protein O-GlcNAcylation.