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Sialylation, a crucial post-translational modification of glycoconjugates, entails the attachment of sialic acid (SA) to the terminal glycans of glycoproteins and glycolipids through a tightly regulated enzymatic process involving various enzymes. This review offers a comprehensive exploration of sialylation within the gut, encompassing its involvement in mucosal protection and its impact on disease progression. The sialylation of mucins and epithelial glycoproteins contributes to the integrity of the intestinal mucosal barrier. Furthermore, sialylation regulates immune responses in the gut, shaping interactions among immune cells, as well as their activation and tolerance. Additionally, the gut microbiota and gut-brain axis communication are involved in the role of sialylation in intestinal health. Altered sialylation patterns have been implicated in various intestinal diseases, including inflammatory bowel disease (IBD), colorectal cancer (CRC), and other intestinal disorders. Emerging research underscores sialylation as a promising avenue for diagnostic, prognostic, and therapeutic interventions in intestinal diseases. Potential strategies such as sialic acid supplementation, inhibition of sialidases, immunotherapy targeting sialylated antigens, and modulation of sialyltransferases have been utilized in the treatment of intestinal diseases. Future research directions will focus on elucidating the molecular mechanisms underlying sialylation alterations, identifying sialylation-based biomarkers, and developing targeted interventions for precision medicine approaches. [Display omitted] [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) are two autoimmune diseases characterised by the production of pathogenic autoreactive antibodies. Their aetiology is poorly understood. Nevertheless, they have been shown to involve several factors, such as infections and epigenetic mechanisms. They also likely involve a physiological process known as glycosylation. Both SLE T cell markers and pSS-associated autoantibodies exhibit abnormal glycosylation. Such dysregulation suggests that defective glycosylation may also occur in B cells, thereby modifying their behaviour and reactivity. This study aimed to investigate B cell subset glycosylation in SLE, pSS and healthy donors and to extend the glycan profile to serum proteins and immunoglobulins. We used optimised lectin-based tests to demonstrate specific glycosylation profiles on B cell subsets that were specifically altered in both diseases. Compared to the healthy donor B cells, the SLE B cells exhibited hypofucosylation, whereas only the pSS B cells exhibited hyposialylation. Additionally, the SLE B lymphocytes had more galactose linked to N-acetylglucosamine or N-acetylgalactosamine (Gal-GlcNAc/Gal-GalNAc) residues on their cell surface markers. Interestingly, some similar alterations were observed in serum proteins, including immunoglobulins. These findings indicate that any perturbation of the natural glycosylation process in B cells could result in the development of pathogenic autoantibodies. The B cell glycoprofile can be established as a preferred biomarker for characterising pathologies and adapted therapeutics can be used for patients if there is a correlation between the extent of these alterations and the severity of the autoimmune diseases. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is one of the most prevalent malignant tumors of the urinary system, accounting for around 2% of all cancer diagnoses and deaths worldwide. Clear cell RCC (ccRCC) is the most prevalent and aggressive histology with an unfavorable prognosis and inadequate treatment. Patients' progression-free survival is considerably improved by surgery; however, 30% of patients develop metastases following surgery. Identifying novel targets and molecular markers for RCC prognostic detection is crucial for more accurate clinical diagnosis and therapy. Glycosylation is a critical post-translational modification (PMT) for cancer cell growth, migration, and invasion, involving the transfer of glycosyl moieties to specific amino acid residues in proteins to form glycosidic bonds through the activity of glycosyltransferases. Most cancers, including RCC, undergo glycosylation changes such as branching, sialylation, and fucosylation. In this review, we discuss the latest findings on the significance of aberrant glycans in the initiation, development, and progression of RCC. The potential biomarkers of altered glycans for the diagnosis and their implications in RCC have been further highlighted. [ABSTRACT FROM AUTHOR]

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells. [ABSTRACT FROM AUTHOR]

The interaction of non-kinase transmembrane glycoprotein CD44 with ligands including hyaluronic acid (HA) is closely related to the occurrence and development of tumors. Changes in CD44 glycosylation can regulate its binding to HA, Siglec-15, fibronectin, TM4SF5, PRG4, FGF2, collagen and podoplanin and activate or inhibit c-Src/STAT3/Twist1/Bmi1, PI3K/AKT/mTOR, ERK/NF-kB/NANOG and other signaling pathways, thereby having a profound impact on the tumor microenvironment and tumor cell fate. However, the glycosylation of CD44 is complex and largely unknown, and the current understanding of how CD44 glycosylation affects tumors is limited. These issues must be addressed before targeted CD44 glycosylation can be applied to treat human cancers. [ABSTRACT FROM AUTHOR]

Streptococcus oralis is an oral commensal and opportunistic pathogen that can enter the bloodstream and cause bacteremia and infective endocarditis. Here, we investigated the mechanisms of S. oralis binding to oral mucins using clinical isolates, isogenic mutants and glycoconjugates. S. oralis bound to both MUC5B and MUC7, with a higher level of binding to MUC7. Mass spectrometry identified 128 glycans on MUC5B, MUC7 and the salivary agglutinin (SAG). MUC7/SAG contained a higher relative abundance of Lewis type structures, including Lewis b/y, sialyl-Lewis a/x and α2,3-linked sialic acid, compared to MUC5B. S. oralis subsp. oralis binding to MUC5B and MUC7/SAG was inhibited by Lewis b and Lacto-N-tetraose glycoconjugates. In addition, S. oralis binding to MUC7/SAG was inhibited by sialyl Lewis x. Binding was not inhibited by Lacto-N-fucopentaose, H type 2 and Lewis x conjugates. These data suggest that three distinct carbohydrate binding specificities are involved in S. oralis subsp. oralis binding to oral mucins and that the mechanisms of binding MUC5B and MUC7 differ. Efficient binding of S. oralis subsp. oralis to MUC5B and MUC7 required the gene encoding sortase A, suggesting that the adhesin(s) are LPXTG-containing surface protein(s). Further investigation demonstrated that one of these adhesins is the sialic acid binding protein AsaA. [ABSTRACT FROM AUTHOR]

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed. [ABSTRACT FROM AUTHOR]

The combination of helium charge transfer dissociation mass spectrometry (He–CTD–MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He–CTD–MS conditions. Results demonstrate that He–CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He–CTD produces informative fragments, including 0,3An and 0,4An ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the α-2,3- versus α-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto- N -tetraose HMOs. In addition to the linkage positions, He–CTD is able to differentiate structural isomers for both sialyllacto- N -tetraoses and lacto- N -fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types 0,2An, 0,2Xn, and 1,5An while preserving most of the labile Neu5Ac and fucose groups. [ABSTRACT FROM AUTHOR]

Background Information: Like most other cell surface proteins, α5β1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. Results: Here, we have established the first comprehensive site‐specific glycan map of α5β1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N‐glycans were predominant at various proportions at most potential N‐glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N‐glycan processing was hindered. Almost all complex N‐glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O‐acetylation of NeuAc residues were observed at certain sites. An O‐linked HexNAc was found in an EGF‐like domain of β1 integrin. The extensive glycan information that results from our study was projected onto a map of α5β1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5β1 integrin. A striking example concerns the involvement of glycan‐binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid‐lectin (GL‐Lect) hypothesis. Conclusion: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5β1 integrin activity and endocytic trafficking. Significance: Glycosylation of α5β1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5β1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins. [ABSTRACT FROM AUTHOR]

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS. [ABSTRACT FROM AUTHOR]

The importance of altered glycosphingolipid (GSL) metabolism is increasingly gaining attention as a characteristic of multiple chronic kidney diseases. Previously, we reported elevated levels of GSLs and neuraminidase (NEU) enzyme activity/expression in the urine or kidney of lupus patients and lupus-prone mice, and demonstrated NEU activity mediates the production of cytokines by lupus-prone mouse primary mesangial cells. This mediation occurs in part through TLR4 and p38/ERK MAPK signalling in response to lipopolysaccharide (LPS) and lupus serum (LS). However, the precise role of NEU1, the most abundant NEU in the kidney, is incompletely known. In this study, we investigated the effect of genetically reduced Neu1 levels in vitro and in vivo. Mesangial cells from non-autoimmune prone Neu1+/− C57BL/6 mice had significantly reduced NEU activity, cytokine expression and cytokine secretion in response to LS and LPS, thereby suggesting reducing Neu1 expression may reduce the inflammatory response in lupus nephritis. Disease was assessed in female B6.SLE1/2/3 lupus-prone mice with genetically reduced levels (Neu1+/−) or wild-type levels (Neu1+/+) of Neu1 from 28 to 44 weeks of age along with aged-matched C57BL/6 controls. Renal disease was unexpectedly mild in all B6.SLE1/2/3 mice despite evidence of systemic disease. B6.SLE1/2/3 Neu1+/− mice exhibited significantly reduced levels of renal NEU1 expression and changes in renal α-2,6 linked sialylated N-glycans compared to the Neu1+/+ or healthy C57BL/6 mice, but measures of renal and systemic disease were similar between the B6.SLE1/2/3 Neu1+/+ and Neu1+/− mice. We conclude that NEU1 is the NEU largely responsible for mediating cytokine release by mesangial cells, at least in vitro, but may not be involved in modulating renal GSL levels in vivo or impact onset of nephritis in lupus-prone mice. However, the effect of reduced NEU1 levels on disease may not be appreciated in the mild disease expression in our colony of B6.SLE1/2/3 mice. The impact of the altered renal sialylated N-glycan levels and potential role of NEU1 with respect to established nephritis (late disease) in lupus-prone mice bears further investigation.

Abstract Individuals infected with the severe acute respiratory syndrome (SARS)‐related coronavirus 2 (SARS‐CoV‐2) develop a critical and even fatal disease, called Coronavirus disease‐19 (COVID‐19), that eventually evolves into acute respiratory distress syndrome. The gravity of the SARS‐CoV‐2 pandemic, the escalating number of confirmed cases around the world, the many unknowns related to the virus mode of action, and the heterogenous outcome of COVID‐19 disease in the population ask for the rapid development of alternative approaches, including repurposing of existing drugs, that may dampen virus infectivity. SARS‐CoV‐2 infects human cells through interaction with sialylated receptors at the surface of epithelial cells, such as angiotensin‐converting enzyme 2 (ACE2). Glycan composition on virus entry receptors has been shown to influence the rate of infection of SARS‐CoV‐2 and spreading of virions has recently been linked to altered lysosomal exocytosis. These processes could concurrently involve the lysosomal system and its glycosidases. We hypothesize that modulating the activity of one of them, the lysosomal sialidase NEU1, could impinge on both the sialylation status of ACE2 and other host receptors as well as the extent of lysosomal exocytosis. Thus NEU1‐controlled pathways may represent therapeutic targets, which could impact on SARS‐CoV‐2 susceptibility, infectivity, and spread.

The Golgi-sialyltransferase ST6Gal1 (βgalactosidase α2,6 sialyltransferase 1), adds the negatively charged sugar, sialic acid, to the terminal galactose of N-glycosylated proteins. Upregulation of ST6Gal1 is observed in many malignancies, and a large body of research has determined that ST6Gal1-mediated α2,6 sialylation impacts cancer hallmarks. ST6Gal1 affects oncogenic behaviors including sustained proliferation, enhanced self-renewal, epithelial-to-mesenchymal transition, invasion, and chemoresistance. However, there are relatively few ST6GaL1 related signaling pathways that are well-established to mediate these biologies: greater delineation of specific targets and signaling mechanisms that are orchestrated by ST6Gal1 is needed. The aim of this review is to provide a summary of our current understanding of select oncogenic signaling pathways and targets affected by ST6Gal1.

Systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are two autoimmune diseases characterised by the production of pathogenic autoreactive antibodies. Their aetiology is poorly understood. Nevertheless, they have been shown to involve several factors, such as infections and epigenetic mechanisms. They also likely involve a physiological process known as glycosylation. Both SLE T cell markers and pSS-associated autoantibodies exhibit abnormal glycosylation. Such dysregulation suggests that defective glycosylation may also occur in B cells, thereby modifying their behaviour and reactivity. This study aimed to investigate B cell subset glycosylation in SLE, pSS and healthy donors and to extend the glycan profile to serum proteins and immunoglobulins. We used optimised lectin-based tests to demonstrate specific glycosylation profiles on B cell subsets that were specifically altered in both diseases. Compared to the healthy donor B cells, the SLE B cells exhibited hypofucosylation, whereas only the pSS B cells exhibited hyposialylation. Additionally, the SLE B lymphocytes had more galactose linked to N-acetylglucosamine or N-acetylgalactosamine (Gal-GlcNAc/Gal-GalNAc) residues on their cell surface markers. Interestingly, some similar alterations were observed in serum proteins, including immunoglobulins. These findings indicate that any perturbation of the natural glycosylation process in B cells could result in the development of pathogenic autoantibodies. The B cell glycoprofile can be established as a preferred biomarker for characterising pathologies and adapted therapeutics can be used for patients if there is a correlation between the extent of these alterations and the severity of the autoimmune diseases.

Streptococcus oralis is an oral commensal and opportunistic pathogen that can enter the bloodstream and cause bacteremia and infective endocarditis. Here, we investigated the mechanisms of S. oralis binding to oral mucins using clinical isolates, isogenic mutants and glycoconjugates. S. oralis bound to both MUC5B and MUC7, with a higher level of binding to MUC7. Mass spectrometry identified 128 glycans on MUC5B, MUC7 and the salivary agglutinin (SAG). MUC7/SAG contained a higher relative abundance of Lewis type structures, including Lewis b/y, sialyl-Lewis a/x and α2,3-linked sialic acid, compared to MUC5B. S. oralis subsp. oralis binding to MUC5B and MUC7/SAG was inhibited by Lewis b and Lacto-N-tetraose glycoconjugates. In addition, S. oralis binding to MUC7/SAG was inhibited by sialyl Lewis x. Binding was not inhibited by Lacto-N-fucopentaose, H type 2 and Lewis x conjugates. These data suggest that three distinct carbohydrate binding specificities are involved in S. oralis subsp. oralis binding to oral mucins and that the mechanisms of binding MUC5B and MUC7 differ. Efficient binding of S. oralis subsp. oralis to MUC5B and MUC7 required the gene encoding sortase A, suggesting that the adhesin(s) are LPXTG-containing surface protein(s). Further investigation demonstrated that one of these adhesins is the sialic acid binding protein AsaA.

BackgroundThe presence of small dense low-density lipoprotein (LDL) is associated with obesity, type II diabetes, and an increased risk for cardiovascular disease. Apolipoprotein C-III (apoC-III) is involved in the formation of small dense LDL, but the exact mechanisms are still not well defined. ApoC-III is a glycosylated apolipoprotein, with 3 major glycoforms: apoC-III0, apoC-III1, and apoC-III2 that contain 0, 1, or 2 molecules of sialic acid, respectively. In our previous work, we reported an association among apoC-III0 and apoC-III1, but not apoC-III2 with fasting plasma triglyceride levels in obesity and type II diabetes.ObjectiveThe goal of this study was to determine the relationship between changes in the major apoC-III glycoforms and small dense LDL levels after dietary interventions.MethodsMass spectrometric immunoassay was performed on fasting plasma samples from 61 subjects who underwent either a high-carbohydrate diet (n = 34) or a weight loss intervention (n = 27).ResultsAfter both dietary interventions, changes in total apoC-III concentrations were associated with changes in LDL peak particle diameter (r = -0.58, P

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties. [ABSTRACT FROM AUTHOR]

Background: Sialic acid–protein interactions are involved in regulating central nervous system immunity; therefore, derangements in sialylation could be involved in neurodegeneration. Objectives: We evaluate the differences in serum transferrin sialylation in prodromal and early‐stage Parkinson's disease (PD), its relation to substantia nigra degeneration, and the risk of phenoconversion to manifest disease. Methods: Sixty treatment‐naive PD patients; 72 polysomnography‐confirmed isolated rapid eye movement sleep behavior disorder (iRBD) patients, that is, patients with prodromal synucleinopathy; and 46 healthy volunteers aged ≥45 years and drinking ≤60 standard drinks per month were included. The proportion of serum low‐sialylated, carbohydrate‐deficient transferrin (CDT) isoforms was assessed using high‐performance liquid chromatography, and the values were adjusted for alcohol intake (CDTadj). Dopamine transporter single‐photon emission computed tomography (DaT‐SPECT) imaging was performed. In iRBD, phenoconversion risk of DaT‐SPECT and CDTadj was evaluated using Cox regression adjusted for age and sex. Results: Median CDTadj was lower in PD (1.1 [interquartile range: 1.0–1.3]%) compared to controls (1.2 [1.1–1.6]%) (P = 0.001). In iRBD, median CDTadj was lower in subjects with abnormal (1.1 [0.9–1.3]%) than normal (1.3 [1.2–1.6]%) DaT‐SPECT (P = 0.005). After a median 44‐month follow‐up, 20% of iRBD patients progressed to a manifest disease. Although iRBD converters and nonconverters did not significantly differ in CDTadj levels (P = 0.189), low CDTadj increased the risk of phenoconversion with hazard ratio 3.2 (P = 0.045) but did not refine the phenoconversion risk associated with abnormal DaT‐SPECT yielding hazard ratio 15.8 (P < 0.001). Conclusions: Decreased serum CDTadj is associated with substantia nigra degeneration in synucleinopathies. iRBD patients with low CDTadj are more likely to phenoconvert to manifest disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Antibodies play a critical role in linking the adaptive immune response to the innate immune system. In humans, antibodies are categorized into five classes, IgG, IgM, IgA, IgE, and IgD, based on constant region sequence, structure, and tropism. In serum, IgG is the most abundant antibody, comprising 75% of antibodies in circulation, followed by IgA at 15%, IgM at 10%, and IgD and IgE are the least abundant. All human antibody classes are post-translationally modified by sugars. The resulting glycans take on many divergent structures and can be attached in an N-linked or O-linked manner, and are distinct by antibody class, and by position on each antibody. Many of these glycan structures on antibodies are capped by sialic acid. It is well established that the composition of the N-linked glycans on IgG exert a profound influence on its effector functions. However, recent studies have described the influence of glycans, particularly sialic acid for other antibody classes. Here, we discuss the role of glycosylation, with a focus on terminal sialylation, in the biology and function across all antibody classes. Sialylation has been shown to influence not only IgG, but IgE, IgM, and IgA biology, making it an important and unappreciated regulator of antibody function. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis presents a complex spectrum of clinical manifestations ranging from asymptomatic microfilariaemic (MF+) to chronic pathology (CP), including lymphedema and elephantiasis. Emerging evidence suggests a link between the physiopathology of filarial infections and antibody properties. Post-translational glycosylation has been shown to play a key role in the modulation of antibodies' effector functions. Here, we investigated the link between total IgG-N-glycosylation patterns and the physiopathology of human lymphatic filariasis using UPLC-FLD/ESI-MS comparison of N-glycan profiles of total IgG purified from endemic normals (EN), MF+, and CP patients. We detected a total of 19 glycans released from all IgG samples. Strikingly, agalactosylated glycan residues were more prominent in EN, whereas sialylation and bisecting GlcNac correlated with asymptomatic infections. While IgG from all three clinical groups expressed high levels of fucosylated residues, significantly lower expressions of afucosylated IgG were seen in MF+ individuals compared to EN and CP. Our data reveal distinct N-linked IgG glycan profiles in EN, MF+, and CP and suggest that IgG galactosylation and sialylation are associated with chronic pathology, whereas agalactosylation correlates with putative immunity. The results also indicate a role for sialylation, fucosylation, and bisecting GlcNac in immune tolerance to the parasite. These findings highlight the link between N-glycosylation and the physiopathology of lymphatic filariasis and open new research avenues for next-generation therapeutic formulations against infectious diseases. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N‐glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi‐OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti‐correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr‐miR‐111 and exhibits two putative miR‐111 binding sites, (3) miR‐111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF‐P. The resulting transcriptional repression of SDK1 led to a downregulation of its co‐expressed miR‐111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation. [ABSTRACT FROM AUTHOR]

Metastasis is considered to be responsible for 90% of cancer-related deaths. Although it is clinically evident that metastatic patterns vary by primary tumor type, the molecular mechanisms underlying the site-specific nature of metastasis are an area of active investigation. One mechanism that has emerged as an important player in this process is glycosylation, or the addition of sugar moieties onto protein and lipid substrates. Glycosylation is the most common post-translational modification, occurring on more than 50% of translated proteins. Many of those proteins are either secreted or expressed on the cell membrane, thereby making glycosylation an important mediator of cell–cell interactions, including tumor-microenvironment interactions. It has been recently discovered that alteration of glycosylation patterns influences cancer metastasis, both globally and in a site-specific manner. This review will summarize the current knowledge regarding the role of glycosylation in the tropism of cancer cells for several common metastatic sites, including the bone, lung, brain, and lymph nodes. [ABSTRACT FROM AUTHOR]

Antibodies play a critical role in linking the adaptive immune response to the innate immune system. In humans, antibodies are categorized into five classes, IgG, IgM, IgA, IgE, and IgD, based on constant region sequence, structure, and tropism. In serum, IgG is the most abundant antibody, comprising 75% of antibodies in circulation, followed by IgA at 15%, IgM at 10%, and IgD and IgE are the least abundant. All human antibody classes are post-translationally modified by sugars. The resulting glycans take on many divergent structures and can be attached in an N-linked or O-linked manner, and are distinct by antibody class, and by position on each antibody. Many of these glycan structures on antibodies are capped by sialic acid. It is well established that the composition of the N-linked glycans on IgG exert a profound influence on its effector functions. However, recent studies have described the influence of glycans, particularly sialic acid for other antibody classes. Here, we discuss the role of glycosylation, with a focus on terminal sialylation, in the biology and function across all antibody classes. Sialylation has been shown to influence not only IgG, but IgE, IgM, and IgA biology, making it an important and unappreciated regulator of antibody function.

The APOE genotype is the most prominent genetic risk factor for the development of late-onset Alzheimer’'s disease (LOAD); however, the underlying mechanisms remain unclear. In the present study, we found that the sialylation profiles of ApoE protein in the human brain are significantly different among the three isoforms, with ApoE2 exhibiting the most abundant sialic acid modification whereas ApoE4 had the least. We further observed that the sialic acid moiety in ApoE2 significantly affected the interaction between ApoE2 and Aβ peptides. The removal of sialic acid in ApoE2 increased the ApoE2 binding affinity for the Aβ17–24 region of Aβ and promoted Aβ fibrillation. These findings provide a plausible explanation for the well-documented differential roles of ApoE isoforms in Aβ pathogenesis. Specifically, compared to the other two isotypes, the higher expression of sialic acid in ApoE2 may contribute to the less potent interaction between ApoE2 and Aβ and ultimately the slower rate of brain Aβ deposition, a mechanism thought to underlie ApoE2-mediated decreased risk for AD. Future studies are warranted to determine whether the differential sialylation in ApoE isoforms may also contribute to some of their other distinct properties, such as their divergent preferences in associations with lipids and lipoproteins, as well as their potential impact on neuroinflammation through modulation of microglial Siglec activity. Overall, our findings lead to the insight that the sialic acid structure is an important posttranslational modification (PTM) that alters ApoE protein functions with relevance for AD.

Microglia are the immune cells in the central nervous system (CNS) and become pro-inflammatory/activated in Alzheimer's disease (AD). Cell surface glycosylation plays an important role in immune cells; however, the N-glycosylation and glycosphingolipid (GSL) signatures of activated microglia are poorly understood. Here, we study comprehensive combined transcriptomic and glycomic profiles using human induced pluripotent stem cells-derived microglia (hiMG). Distinct changes in N-glycosylation patterns in amyloid-β oligomer (AβO) and LPS-treated hiMG were observed. In AβO-treated cells, the relative abundance of bisecting N-acetylglucosamine (GlcNAc) N-glycans decreased, corresponding with a downregulation of MGAT3. The sialylation of N-glycans increased in response to AβO, accompanied by an upregulation of genes involved in N-glycan sialylation (ST3GAL4 and 6). Unlike AβO-induced hiMG, LPS-induced hiMG exhibited a decreased abundance of complex-type N-glycans, aligned with downregulation of mannosidase genes (MAN1A1, MAN2A2, and MAN1C1) and upregulation of ER degradation related-mannosidases (EDEM1-3). Fucosylation increased in LPS-induced hiMG, aligned with upregulated fucosyltransferase 4 (FUT4) and downregulated alpha-L-fucosidase 1 (FUCA1) gene expression, while sialofucosylation decreased, aligned with upregulated neuraminidase 4 (NEU4). Inhibition of sialyation and fucosylation in AβO- and LPS-induced hiMG alleviated pro-inflammatory responses. However, the GSL profile did not exhibit significant changes in response to AβO or LPS activation. AβO- and LPS- specific glycosylation changes could contribute to impaired microglia function, highlighting glycosylation pathways as potential therapeutic targets for AD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Additional Declarations: No competing interests reported.

Purpose: The purpose of this study was to detect the distribution of different lectin receptors in primary cancer cells as well as in the case of metastasis, as these biomolecules can potentially predict cancer development in certain tissues and systems., Methods: To detect lectin receptors in tumors, the authors used conjugates of lectins purified by affinity chromatography with peroxidase, and studied their localization in paraffin sections of 12 cases of primary cancer and 33 of its metastases., Result: In primary cancers and their metastases, there is a distinct mosaicity in the histotopography of individual lectins, especially peanut and soybean lectin. Mosaicity increases in metastases, which corresponds to the increase in malignancy of tumour cells. Detected cases of metastases with a decrease in mosaicity may be a sign of their monoclonality or a decrease in their malignancy. The study of lectins in the cells of cancer metastases and their comparison with the primary tumour and with each other suggests that in metastases, as a rule, not only signs of malignancy increase, but also the ability of cells to adhere and migrate. Thus, in the process of tumour growth and metastasis, there is a selection of clones of cells that are more prone to the development of new metastases., Conclusions: It has been revealed that the growth of a primary immature tumour from the epithelium and its metastases is accompanied by sialylation of the surface of tumour cells, which leads to the fact that tumour cells are not recognized by the system of mononuclear phagocytes and this, naturally, contributes to the progression of tumour growth.

Sialidases (or neuraminidases) catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly the removal of the terminal Sia on glycans (desialylation) of either glycoproteins or glycolipids. Therefore, sialidases can modulate the functionality of the target glycoprotein or glycolipid and are involved in various biological pathways in health and disease. In mammalian cells, there are four kinds of sialidase, which are Neu1, Neu2, Neu3, and Neu4, based on their subcellular locations and substrate specificities. Neu1 is the lysosomal sialidase, Neu2 is the cytosolic sialidase, Neu3 is the plasma membrane-associated sialidase, and Neu4 is found in the lysosome, mitochondria, and endoplasmic reticulum. In addition to specific subcellular locations, sialidases can translocate to different subcellular localizations within particular cell conditions and stimuli, thereby participating in different cellular functions depending on their loci. Lysosomal sialidase Neu1 can translocate to the cell surface upon cell activation in several cell types, including immune cells, platelets, endothelial cells, and epithelial cells, where it desialylates receptors and thus impacts receptor activation and signaling. On the other hand, cells secrete sialidases upon activation. Secreted sialidases can serve as extracellular sialidases and cause the desialylation of both extracellular glycoproteins or glycolipids and cell surface glycoproteins or glycolipids on their own and other cells, thus playing roles in various biological pathways as well. This review discusses the recent advances and understanding of sialidase translocation in different cells and secretion from different cells under different conditions and their involvement in physiological and pathological pathways., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity. Key points: • Valine feeding can mitigate the negative impacts of ammonia on CHO cell growth. • Valine addition assists the ammonia removal mechanism by enriching the TCA cycle. • Ammonia is removed from the media through alanine excretion in valine-fed culture. [ABSTRACT FROM AUTHOR]

Background: Hereditary angioedema (HAE) is a rare disease with heterogeneous clinical symptoms. It is vitally important to predict whether an HAE patient will develop severe symptoms in clinical practice, but there are currently no predictive biomarkers for HAE stratification. Plasma N‐glycomes are disease‐specific and have great potential for the discovery of non‐invasive biomarkers. In this study, we profiled the plasma N‐glycome of HAE patients from two independent cohorts to identify candidate biomarkers. Methods: Linkage‐specific sialylation derivatization combined with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry detection and automated data processing was employed to analyze the plasma N‐glycome of two independent type‐1 HAE cohorts. Results: HAE patients had abnormal glycan complexity, galactosylation, and α2,3‐ and α2,6‐linked sialylation compared to healthy controls (HC). The classification models based on dysregulated glycan traits could successfully discriminate between HAE and HC with area under the curves (AUCs) being greater than 0.9. Some of the aberrant glycans showed response to therapy. Moreover, we identified a series of glycan traits with strong associations with the occurrence of laryngeal or gastrointestinal angioedema or disease severity score. Predictive models based on these traits could be used to predict disease severity (AUC > 0.9). These results were replicated in an independent cohort. Conclusions: We reported the full plasma N‐glycomic signature of HAE for the first time, and identified potential biomarkers. These findings may play a critical role in predicting disease severity and guide the treatment of HAE in clinical practice. Further protein‐specific and prospective studies are needed to validate our findings. [ABSTRACT FROM AUTHOR]

UDP-N-Acetyl glucosamine-2 epimerase/N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE myopathy (GNEM) characterized by adult-onset muscle weakness and degeneration. However, recent studies propose alternate roles of GNE in other cellular processes beside sialic acid biosynthesis, particularly interaction of GNE with α-actinin 1 and 2. Lack of appropriate model system limits drug and treatment options for GNEM as GNE knockout was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle myoblast cell-based model system carrying one single Gne allele where GNE gene is knocked out at exon-3 using AAV mediated SEPT homology recombination (SKM-GNEHz). The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the heterozygous GNE knockout cells revealed altered cytoskeletal organization with disrupted actin filament. Further, we observed increased levels of RhoA leading to reduced cofilin activity and causing reduced F-actin polymerization. The disturbed signaling cascade resulted in reduced migration of SKM-GNEHz cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell-based system offers great potential in understanding pathomechanism and target identification for GNEM. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation, particularly sialylation is considered a significant characteristic of malignancy. Aberrant sialylation has been connected with several neoplastic disorders. The role of abnormal sialylation in oral cancer blood, on the other hand, is still in its early stages. The present study focussed to examine relative quantification of mRNA expression levels of Dolichyl-Phosphate N-Acetylglucosaminephosphotransferase 1 (DPAGT1) encoding enzyme that catalyzes the first step in the dolichol-linked oligosaccharide pathway for glycosylation, Beta-1,4-Mannosyl-Glycoprotein 4-Beta-N-Acetylglucosaminyltransferase (MGAT3) and Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase (MGAT5) mediated N-glycosylation, selected α2, 3, α2, 6 sialyltransferases families and sialidase in thirty human oral cancer blood and twelve control samples by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method. The results indicated significant upregulation of MGAT3, ST3 Beta-Galactoside Alpha-2, 3-Sialyltransferase 1 (ST3GAL1), ST6 N-Acetylgalactosaminide Alpha-2, 6-Sialyltransferase 1(ST6GALNAC1), and Neuraminidase 3 (NEU3) compared to healthy controls. In terms of habits, tobacco chewing and smoking, high expression levels of ST3GAL1 and ST6 Beta-Galactoside Alpha-2, 6-Sialyltransferase 1 (ST6GAL1) were observed. Although, MGAT3 and NEU3 elevated expression were statistically significant in patients who had no habits. The expressions of DPAGT1, MGAT3, MGAT5, ST3GAL1, ST3 Beta-Galactoside Alpha-2, 3-Sialyltransferase 6 (ST3GAL6), ST6GALNAC1, and NEU3 were also linked to the disease severity and progression. Hence, this study demonstrated the significant role of elevated mRNA levels of ST3GAL1, ST6GALNAC1, and NEU3 in peripheral blood mononucleated cells of oral cancer disease progression.

BackgroundHereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack.MethodsSialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays.ResultsThe proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis.ConclusionWe report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

Background: Hereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP- N -acetyl-glucosamine-2-epimerase/ N -acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack. Methods: Sialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays. Results: The proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis. Conclusion: We report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis. [ABSTRACT FROM AUTHOR]

Abnormal sialylation is a distinctive feature of human hepatocellular carcinoma (HCC) and is closely related to its malignant properties. Exosomes have characteristic protein and lipid composition; however, the results concerning glycoprotein composition and glycosylation are scarce. In this study, liquid chromatography–tandem mass spectrometry (LC‐MS/MS) identified multiple microvesicle‐related sialylated proteins including CD63, a classic marker of exosomes. The silencing of α2,6‐sialyltransferase I (ST6Gal‐I) significantly reduced the levels of α2,6‐sialylated glycoconjugates on CD63 and the surface of HCC‐derived exosomes (HCC‐exo). And surface glycoconjugates play important roles in exosomes biogenesis and in their interaction with other cells. Compared to exosomes derived from naive HCC cells, α2,6‐sialylation degradation abolished both the proliferation‐promoting and migration‐promoting effects of HCC‐exo. Further analysis revealed that the Akt/GSK‐3β or JNK1/2 signaling mediates HCC‐exo‐mediated proliferation in HCC cells, while ST6Gal‐I silencing deactivated this pathway. These findings suggest that a loss of α2,6‐sialylation decreases HCC progression through the loss of cancer cell‐derived exosomes; furthermore, it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes. [ABSTRACT FROM AUTHOR]

It has been well documented that the terminal sugars of Fc glycans can play a critical role in the safety and efficacy of therapeutic mAbs. However, many of the effects of highly heterogeneous Fc glycan structures have yet to be fully characterized. Different glycosylation patterns can affect Fc‐dependent activities, such as the ability of mAbs to bind Fcγ receptors on the effector cell surface, which is critical to immune effector functions, such as antibody‐dependent cellular cytotoxicity (ADCC). Previous studies on the impact of sialic acid in the Fc glycan on ADCC have not resulted in consistent conclusions. In our study, we tested sialic acid‐enriched species from a chimeric murine/human kappa light chain IgG1 (mAb1) with known Fcγ receptor IIIa binding and ADCC activities. These enriched species contained up to a fourfold increase in sialic acid‐containing glycans relative to the typical levels present in therapeutic mAbs, along with other attributes such as oxidized and deamidated species. The ADCC analysis of sialylated and asialo mAb1 provided herein shows evidence that sialic acids have little or no impact on ADCC activity. Altogether, our results highlight the value of novel glycan engineering strategies in designing therapeutic mAbs with high‐quality attributes and in improving production process controls. [ABSTRACT FROM AUTHOR]

Sialylation of glycoproteins, including integrins, is crucial in various cancers and diseases such as immune disorders. These modifications significantly impact cellular functions and are associated with cancer progression. Sialylation, catalyzed by specific sialyltransferases (STs), has traditionally been considered to be regulated at the mRNA level. Recent research has expanded our understanding of sialylation, revealing ST activity changes beyond mRNA level variations. This includes insights into COPI vesicle formation and Golgi apparatus maintenance and identifying specific target proteins of STs that are not predictable through recombinant enzyme assays. This review summarizes that Golgi-associated pathways largely influence the regulation of STs. GOLPH3, GORAB, PI4K, and FAK have become critical elements in sialylation regulation. Some STs have been revealed to possess specificity for specific target proteins, suggesting the presence of additional, enzyme-specific regulatory mechanisms. This study enhances our understanding of the molecular interplay in sialylation regulation, mainly focusing on the role of integrin and FAK. It proposes a bidirectional system where sialylations might influence integrins and vice versa. The diversity of STs and their specific linkages offer new perspectives in cancer research, potentially broadening our understanding of cellular mechanisms and opening avenues for new therapeutic approaches in targeting sialylation pathways. • Epigenetic and transcriptional factors influence ST gene expressions. • GOLPH3 and GORAB regulate sialylation through their roles in COPI vesicles. • FAK modulates the stability of PI4KIIα, influencing N-glycan sialylation. • ST6GAL1 and the ST3GAL family have distinct target substrates. [ABSTRACT FROM AUTHOR]

Leqi Zhou,*,1,2 Sen Zhang,*,1,2 Xia Zou,3 Jishun Lu,3 Xiao Yang,1,2 Zhijue Xu,3 Aidong Shan,3 Wenjuan Jia,3 Feng Liu,3 Xialin Yan,1,2 Hao Su,1,2 Tao Liang,3 Minhua Zheng,1,2 Yan Zhang,3 Bo Feng1,21Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, Shanghai, People’s Republic of China*These authors contributed equally to this workBackground: Colorectal cancer (CRC) is one of the most frequent malignancies of the digestive system. Elevated expression of β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in multiple cancers. But the mechanism of how ST6GAL1 might affect cancer cells remains to be clarified. Our previous study recognized intercellular adhesion molecule-1(ICAM-1) as a probable substrate of ST6GAL1 through mass spectrometry (MS) analysis. ICAM-1 is related to tumor metastasis in various cancers.Methods: First, ST6GAL1 was overexpressed and knocked down to perform transwell and wound healing assays, and the results were further confirmed in vivo. Based on the results of MS, GO and KEGG analysis were applied to reveal the connection between ST6GAL1 and ICAM-1. Immunoblot and tissue microarrays were administered to investigate the expression of ICAM-1 in different stages of CRC. Next, PCR, lectin precipitation and cycloheximide (CHX) were used to demonstrate the mechanism of ST6GAL1 on ICAM-1. Moreover, we investigated the sialylation on soluble ICAM in serum and its connection to tumor staging.Results: Overexpression of ST6GAL1 inhibited the migratory ability, while knockdown of ST6GAL1 cells had the reverse effect. Moreover, nude mice injected with ST6GAL1-knockdown cells harvested more liver metastases. Based on the GO and KEGG analysis, data from TCGA database showed a positive correlation between ST6GAL1 and ICAM-1. ICAM-1 also demonstrated a significant decrease in stage III/IV compared with stage I/II tumors. Our results revealed that ST6GAL1 could increase the stability of ICAM-1 through sialylation but had little influence on transcriptional level. Additionally, results of serum lectin precipitation revealed a correlation between the level of sialylation on soluble ICAM and CRC staging.Conclusion: This study illustrated that ST6GAL1 inhibited the metastatic ability of CRC by stabilizing ICAM-1 via sialylation and demonstrated a correlation between CRC staging and the sialylation on soluble ICAM-1 in serum.Keywords: colorectal cancer, β-galactoside α2, 6-sialyltranferase 1, ST6GAL1, intercellular adhesion molecule-1, ICAM-1, sialylation, metastasis

Objective: Sialidases can play an important role in atherosclerosis development due to modification of low-density lipoproteins (LDL). It is known that desialylated LDLs are associated with atherosclerosis development. However, the information about factors leading to desialylation of LDL needs clarifying. The creation of an appropriate model object could help to understand factors related to desialylation of LDL. The aim of this study was to test the possibility to change sialylation of lipoproteins in healthy mice upon an injection of immobilized sialidase. Methods: The control group of C57BL6 mice (n = 48) was treated by a single injection of saline, while the experimental group (n = 48) received Vibrio cholerae sialidase conjugated with mouse IgG. Mice were terminated at fixed periods: before and after a single injection (one to seven days). LDL was isolated from serum by ultracentrifugation. The content of sialic acid was determined according to Warren’s method. Lipids of serum were measured by commercial kits. Results: A significant decrease in LDL sialic acid by 30% was detected up to five days after the sialidase injection. Additionally, serum levels of triglycerides, total cholesterol, and HDL-cholesterol in experimental mice did not differ compared with wild-type control mice. Conclusions: A new approach to study the role of sialidase as a proatherogenic factor in vivo was established.

BackgroundBiomarkers are needed for patient stratification between benign thyroid nodules (BTN) and thyroid cancer (TC) and identifying metastasis in TC. Though plasma N-glycome profiling has shown potential in the discovery of biomarkers and can provide new insight into the mechanisms involved, little is known about it in TC and BTN. Besides, several studies have indicated associations between abnormal glycosylation and TC. Here, we aimed to explore plasma protein N-glycome of a TC cohort with regard to their applicability to serve as biomarkers.MethodsPlasma protein N-glycomes of TC, BTN, and matched healthy controls (HC) were obtained using a robust quantitative strategy based on MALDI-TOF MS and included linkage-specific sialylation information.ResultsPlasma N-glycans were found to differ between BTN, TC, and HC in main glycosylation features, namely complexity, galactosylation, fucosylation, and sialylation. Four altered glycan traits, which were consecutively decreased in BTN and TC, and classification models based on them showed high potential as biomarkers for discrimination between BTN and TC (“moderately accurate” to “accurate”). Additionally, strong associations were found between plasma N-glycans and lymph node metastasis in TC, which added the accuracy of predicting metastasis before surgery to the existing method.ConclusionsWe comprehensively evaluated the plasma N-glycomic changes in patients with TC or BTN for the first time. We determined several N-glycan biomarkers, some of them have potential in the differential diagnosis of TC, and the others can help to stratify TC patients to low or high risk of lymph node metastasis. The findings enhanced the understanding of TC.

• ST6Gal-I promotes Aβ peptide aggregation and then accelerates the process of AD. • Gangliosides are essential for the development of the central nervous system. • Sialylated HMOs could improve the cognitive benefits. • CD33 is highly expressed in the brain of AD patients. Sialylation, one of the most common and complex modes of glycosylation, corresponds with the development of the infant brain and nervous system. The most prevalent neurodegenerative disease is Alzheimer's disease (AD), which is mainly characterized by cognitive decline and behavioral disorders. However, the relationship between sialylation and AD occurrence is poorly understood. In this article, we reviewed the role of sialylation on the occurrence and development of AD, then discussed the value of sialylation modification for AD diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy. [ABSTRACT FROM AUTHOR]

N -glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N -acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N -glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N -glycosylation and superior pharmacokinetic properties. [ABSTRACT FROM AUTHOR]

In skeletal muscles, the sialic acids have a great signifi cance for their functional maintenance and proper structural organization. Our work described the expressions of St3gal, St6gal and St6galnac sialyltransferases specifi c for glycoproteins in mouse skeletal muscles and murine C2C12 myotubes. Lectin histochemistry, cytochemistry and lectin blot were used to demonstrate the membrane localization and the electrophoretic profi les of a-2,3- and a-2,6-sialylated glycoproteins. The expression levels of sialyltransferases were analysed by real time RT-PCR and western blot. The enzymes St6gal2 and St6galnac1 were not expressed in skeletal muscle tissue and C2C12 myotubes. In both experimental groups, mRNAs of the St3gal family prevailed over the mRNA expressions of the St6gal and St6galnac families. The profi les of sialyltransferase expressions showed differences between the two experimental groups, illustrated by the absence of expressions of the mRNA for the St3gal6 and St6galnac3 genes in the C2C12 cell samples and by the diff erent shares of the enzymes St3gal3 and St3gal4 in both experimental groups. The diff erent patterns of enzyme expressions in both experimental groups corresponded with differences between their a-2,3- and a-2,6-sialylated glycoprotein profi les. These results could be a useful addendum to the knowledge concerning the glycosylation of the skeletal muscle tissue. [ABSTRACT FROM AUTHOR]

Background: Biomarkers are needed for patient stratification between benign thyroid nodules (BTN) and thyroid cancer (TC) and identifying metastasis in TC. Though plasma N -glycome profiling has shown potential in the discovery of biomarkers and can provide new insight into the mechanisms involved, little is known about it in TC and BTN. Besides, several studies have indicated associations between abnormal glycosylation and TC. Here, we aimed to explore plasma protein N -glycome of a TC cohort with regard to their applicability to serve as biomarkers. Methods: Plasma protein N -glycomes of TC, BTN, and matched healthy controls (HC) were obtained using a robust quantitative strategy based on MALDI-TOF MS and included linkage-specific sialylation information. Results: Plasma N -glycans were found to differ between BTN, TC, and HC in main glycosylation features, namely complexity, galactosylation, fucosylation, and sialylation. Four altered glycan traits, which were consecutively decreased in BTN and TC, and classification models based on them showed high potential as biomarkers for discrimination between BTN and TC ("moderately accurate" to "accurate"). Additionally, strong associations were found between plasma N -glycans and lymph node metastasis in TC, which added the accuracy of predicting metastasis before surgery to the existing method. Conclusions: We comprehensively evaluated the plasma N -glycomic changes in patients with TC or BTN for the first time. We determined several N -glycan biomarkers, some of them have potential in the differential diagnosis of TC, and the others can help to stratify TC patients to low or high risk of lymph node metastasis. The findings enhanced the understanding of TC. [ABSTRACT FROM AUTHOR]

• Media supplements MGU (Mg, Gal, Uridine) increase Mab galactosylation from NS0 and CHO cells. • MGU increases galactosylation & sialylation of the NSO Mab by enhanced Galα1−3Gal and Neu5Ac. • Neu5Gc is the predominant sialic acid in Mabs from NS0 cells but negligible in Mabs from CHO cells. • Fetal bovine serum resulted in decreased Neu5Gc and enhanced Neu5Ac sialylation from NS0 cells. The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1−3 Gal that may be immunogenic. We have looked at the effects of a commonly used media supplements of manganese, galactose and uridine (MGU) on Mab production from CHO and NS0 cells in enhancing galactosylation and sialylation as well as the generation of these non-human glycan epitopes. In the absence of the MGU supplement, the humanized IgG1 antibody (Hu1D10) produced from NS0 cells showed a low level of mono- and di-sialylated structures (SI:0.09) of which 75 % of sialic acid was Neu5Gc. The chimeric human-llama Mab (EG2-hFc) produced from CHO cells showed an equally low level of sialylation (SI: 0.12) but the Neu5Gc content of sialic acid was negligible (<3%). Combinations of the MGU supplements added to the production cultures resulted in a substantial increase in the galactosylation of both Mabs (up to GI:0.78 in Hu1D10 and 0.81 in EG2-hFc). However, the effects on sialylation differed between the two Mabs. We observed a slight increase in sialylation of the EG2-hFc Mab by a combination of MG but it appeared that one of the components (uridine) was inhibitory to sialylation. On the other hand, MG or MGU increased sialylation of Hu1D10 substantially (SI:0.72) with an increase that could be attributed predominantly to the formation of Neu5Ac rather than Neu5Gc. The increased level of galactosylation observed with MG or MGU was attributed to an activation of the galactosyl transferase enzymes through enhanced intracellular levels of UDP-Gal and the availability of Mn2+ as an enzymic co-factor. However, this effect not only increased the desirable beta 1−4 Gal linkage to GlcNAc but unfortunately in NS0 cells increased the formation of Galα1−3 Gal which was shown to increase x3 in the presence of combinations of the MGU supplements. Supplementation of media with fetal bovine serum (FBS) increased the availability of free Neu5Ac which resulted in a significant increase in the sialylation of Hu1D10 from NS0 cells. This also resulted in a significant decrease in the proportion of Neu5Gc in the measured sialic acid from the Mab. [ABSTRACT FROM AUTHOR]