Presence of heavy chain-hyaluronan/pentraxin 3 (HC-HA/PTX3) complex in human umbilical cord.

The heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (HC-HA/PTX3) complex is formed by tumor necrosis factor-stimulated gene-6 (TSG-6) catalyzing the covalent (ester bond) transfer of HC1 from inter-α-trypsin inhibitor (IαI) to HA followed by tight binding of PTX3. The presence of such a complex has been found in human amniotic membrane (AM) and is considered to be a major matrix component responsible for its anti­inflammatory and anti­scarring properties to promote regenerative healing. Because the therapeutic potentials of AM and umbilical cord (UC) are similar, we herein evaluated whether human UC also contains HC-HA/PTX3. Immunostaining of UC cross-sections showed abundant PTX3, HC1, HA, TSG-6, and bikunin. Western blot analysis suggested the presence of HC1 complex bound via a NaOH-sensitive bond and tightly bound to PTX3 multimer in UC and AM extracts but not in chorion and placenta extracts. HC-HA/PTX3 was purified from UC extract by successive runs of density gradient ultracentrifugation and verified the presence of HC1 but not HC2 or HC3 based on western blot analysis. These results suggest the presence of HC-HA/PTX3 complex in UC is similar to AM.

Potential Targeting Mechanisms for Bone-Directed Therapies.

Bone development is characterized by complex regulation mechanisms, including signal transduction and transcription factor-related pathways, glycobiological processes, cellular interactions, transportation mechanisms, and, importantly, chemical formation resulting from hydroxyapatite. Any abnormal regulation in the bone development processes causes skeletal system-related problems. To some extent, the avascularity of cartilage and bone makes drug delivery more challenging than that of soft tissues. Recent studies have implemented many novel bone-targeting approaches to overcome drawbacks. However, none of these strategies fully corrects skeletal dysfunction, particularly in growth plate-related ones. Although direct recombinant enzymes (e.g., Vimizim for Morquio, Cerezyme for Gaucher, Elaprase for Hunter, Mepsevii for Sly diseases) or hormone infusions (estrogen for osteoporosis and osteoarthritis), traditional gene delivery (e.g., direct infusion of viral or non-viral vectors with no modifications on capsid, envelope, or nanoparticles), and cell therapy strategies (healthy bone marrow or hematopoietic stem cell transplantation) partially improve bone lesions, novel delivery methods must be addressed regarding target specificity, less immunogenicity, and duration in circulation. In addition to improvements in bone delivery, potential regulation of bone development mechanisms involving receptor-regulated pathways has also been utilized. Targeted drug delivery using organic and inorganic compounds is a promising approach in mostly preclinical settings and future clinical translation. This review comprehensively summarizes the current bone-targeting strategies based on bone structure and remodeling concepts while emphasizing potential approaches for future bone-targeting systems.

Revisiting legume lectins: Structural organization and carbohydrate-binding properties.

Legume lectins are a diverse family of carbohydrate-binding proteins that share significant similarities in their primary, secondary, and tertiary structures, yet exhibit remarkable variability in their quaternary structures and carbohydrate-binding specificities. The tertiary structure of legume lectins, characterized by a conserved ß-sandwich fold, provides the scaffold for the formation of a carbohydrate-recognition domain (CRD) responsible for ligand binding. The structural basis for the binding is similar between members of the family, with key residues interacting with the sugar through hydrogen bonds, hydrophobic interactions, and van der Waals forces. Variability in substructures and residues within the CRD are responsible for the large array of specificities and enable legume lectins to recognize diverse sugar structures, while maintaining a consistent structural fold. Therefore, legume lectins can be classified into several specificity groups based on their preferred ligands, including mannose/glucose-specific, N-acetyl-d-galactosamine/galactose-specific, N-acetyl-d-glucosamine-specific, l-fucose-specific, and α-2,3 sialic acid-specific lectins. In this context, this review examined the structural aspects and carbohydrate-binding properties of representative legume lectins and their specific ligands in detail. Understanding the structure/binding relationships of lectins continues to provide valuable insights into their biological roles, while also assisting in the potential applications of these proteins in glycobiology, diagnostics, and therapeutics.

Bacterial glycoengineering: Cell-based and cell-free routes for producing biopharmaceuticals with customized glycosylation.

Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

Analysis of Mucin-Domain Glycoproteins Using Mass Spectrometry.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides.

Single-cell transcriptomic analysis to identify endomembrane regulation of metalloproteins and motor proteins in autoimmunity.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Cell-based glycoengineering of extracellular vesicles through precise genome editing.

Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a "simple cell" strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.

Transmembrane protein TMEM230, regulator of metalloproteins and motor proteins in gliomas and gliosis.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

The pleiotropic CLEC10A: implications for harnessing this receptor in the tumor microenvironment.

INTRODUCTION: CLEC10A is a C-type lectin receptor that specifically marks the conventional dendritic cell subsets two and three (cDC2 and DC3). It has a unique recognition profile of glycan antigens, with terminal N-Acetylgalactosamine residues that are frequently present in the tumor microenvironment. Even though CLEC10A expression allows for precise targeting of cDC2 and DC3 for the treatment of cancer, CLEC10A signaling has also been associated with anti-inflammatory responses that would promote tumor growth. AREAS COVERED: Here, we review the potential benefits and drawbacks of CLEC10A engagement in the tumor microenvironment. We discuss the CLEC10A-mediated effects in different cell types and incorporate the pleiotropic effects of IL-10, the main anti-inflammatory response upon CLEC10A binding. EXPERT OPINION: To translate this to a successful CLEC10A-mediated immunotherapy with limited tumor-promoting capacities, finding the right ligand presentation and adjuvant combination will be key.

Regulation of intracellular activity of N-glycan branching enzymes in mammals.

Most proteins in the secretory pathway are glycosylated, and N-glycans are estimated to be attached to over 7000 proteins in humans. As structural variation of N-glycans critically regulates the functions of a particular glycoprotein, it is pivotal to understand how structural diversity of N-glycans is generated in cells. One of the major factors conferring structural variation of N-glycans is the variable number of N-acetylglucosamine branches. These branch structures are biosynthesized by dedicated glycosyltransferases, including GnT-III (MGAT3), GnT-IVa (MGAT4A), GnT-IVb (MGAT4B), GnT-V (MGAT5), and GnT-IX (GnT-Vb, MGAT5B). In addition, the presence or absence of core modification of N-glycans, namely, core fucose (included as an N-glycan branch in this manuscript), synthesized by FUT8, also confers large structural variation on N-glycans, thereby crucially regulating many protein-protein interactions. Numerous biochemical and medical studies have revealed that these branch structures are involved in a wide range of physiological and pathological processes. However, the mechanisms regulating the activity of the biosynthetic glycosyltransferases are yet to be fully elucidated. In this review, we summarize the previous findings and recent updates regarding regulation of the activity of these N-glycan branching enzymes. We hope that such information will help readers to develop a comprehensive overview of the complex system regulating mammalian N-glycan maturation.

Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast.

The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.

Uncloaking the viral glycocalyx: How do viruses exploit glycoimmune checkpoints?

The surfaces of cells and enveloped viruses alike are coated in carbohydrates that play multifarious roles in infection and immunity. Organisms across all kingdoms of life make use of a diverse set of monosaccharide subunits, glycosidic linkages, and branching patterns to encode information within glycans. Accordingly, sugar-patterning enzymes and glycan binding proteins play integral roles in cell and organismal biology, ranging from glycoprotein quality control within the endoplasmic reticulum to lymphocyte migration, coagulation, inflammation, and tissue homeostasis. Unsurprisingly, genes involved in generating and recognizing oligosaccharide patterns are playgrounds for evolutionary conflicts that abound in cross-species interactions, exemplified by the myriad plant lectins that function as toxins. In vertebrates, glycans bearing acidic nine-carbon sugars called sialic acids are key regulators of immune responses. Various bacterial and fungal pathogens adorn their cells in sialic acids that either mimic their hosts' or are stolen from them. Yet, how viruses commandeer host sugar-patterning enzymes to thwart immune responses remains poorly studied. Here, we review examples of viruses that interact with sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immune cell receptors that regulate toll-like receptor signaling and govern glycoimmune checkpoints, while highlighting knowledge gaps that merit investigation. Efforts to illuminate how viruses leverage glycan-dependent checkpoints may translate into new clinical treatments that uncloak viral antigens and infected cell surfaces by removing or masking immunosuppressive sialoglycans, or by inhibiting viral gene products that induce their biosynthesis. Such approaches may hold the potential to unleash the immune system to clear long intractable chronic viral infections.

Tumor glucose metabolism and the T cell glycocalyx: implication for T cell function.

The T cell is an immune cell subset highly effective in eliminating cancer cells. Cancer immunotherapy empowers T cells and occupies a solid position in cancer treatment. The response rate, however, remains relatively low (<30%). The efficacy of immunotherapy is highly dependent on T cell infiltration into the tumor microenvironment (TME) and the ability of these infiltrated T cells to sustain their function within the TME. A better understanding of the inhibitory impact of the TME on T cells is crucial to improve cancer immunotherapy. Tumor cells are well described for their switch into aerobic glycolysis (Warburg effect), resulting in high glucose consumption and a metabolically distinct TME. Conversely, glycosylation, a predominant posttranslational modification of proteins, also relies on glucose molecules. Proper glycosylation of T cell receptors influences the immunological synapse between T cells and tumor cells, thereby affecting T cell effector functions including their cytolytic and cytostatic activities. This review delves into the complex interplay between tumor glucose metabolism and the glycocalyx of T cells, shedding light on how the TME can induce alterations in the T cell glycocalyx, which can subsequently influence the T cell's ability to target and eliminate tumor cells.

LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

N-glycosylation by Mgat5 imposes a targetable constraint on immune-mediated tumor clearance.

The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of ß1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.

Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus.

Objectives: Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Methods: Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Results: Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Conclusion: Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE.

Glycoproteomics: Charting new territory in mass spectrometry and glycobiology.

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.