Search Results (22)

Galectin-3 (Gal-3) is a β-galactoside-binding lectin that mediates diverse signaling events in multiple cell types, including immune cells. It is also a prognostic indicator for multiple clinically important disorders, including cardiovascular disease. Gal-3 binds to cell surface glycans to form lattices that modulate surface receptor signaling and internalization. However, the tissue-specific regulation of Gal-3 surface expression remains poorly understood. Here, we review evidence for the involvement of Gal-3 in cell surface signaling, intranuclear events, and intracellular trafficking. Our focus will be on the O-GlcNAc modification as a regulator of Gal-3 biosynthesis, non-canonical secretion, and recycling. We argue that the nutrient-driven cytoplasmic hexosamine biosynthetic pathway (HBP) and endomembrane transport mechanisms generate unique pools of nucleotide sugars. The differing levels of nucleotide sugars in the cytosol, endoplasmic reticulum (ER), and Golgi apparatus generate differential thresholds for the responsiveness of O-GlcNAc cycling, N- and O-linked glycan synthesis/branching, and glycolipid synthesis. By regulating Gal-3 synthesis and non-canonical secretion, O-GlcNAc cycling may serve as a nexus constraining Gal-3 cell surface expression and lattice formation. This homeostatic feedback mechanism would be critical under conditions where extensive glycan synthesis and branching in the endomembrane system and on the cell surface are maintained by elevated hexosamine synthesis. Thus, O-GlcNAc cycling and Gal-3 synergize to regulate Gal-3 secretion and influence cellular signaling. In humans, Gal-3 serves as an early-stage prognostic indicator for heart disease, kidney disease, viral infection, autoimmune disease, and neurodegenerative disorders. Since O-GlcNAc cycling has also been linked to these pathologic states, exploring the interconnections between O-GlcNAc cycling and Gal-3 expression and synthesis is likely to emerge as an exciting area of research. Full article

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions of infections and millions of deaths globally. Although vaccination campaigns are mitigating the pandemic, emerging viral variants continue to pose challenges. The spike (S) protein of SARS-CoV-2 plays a critical role in viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, making both proteins essential targets for therapeutic and vaccine development. The glycosylation of these proteins influences their structure and function. This underscores the need for detailed site-specific glycoproteomic analysis. In this study, we characterized the N- or O-glycosylation profiles of the recombinant receptor-binding domain (RBD) of spike protein and ACE2 proteins expressed from Expi293F cells, as well as the S2 subunit of spike protein expressed in plant (N. benthamiana) cells. Using a high-resolution Orbitrap Eclipse Tribrid mass spectrometer equipped with the Ultimate 3000 RSLCnano and I-GPA (Integrated GlycoProteome Analyzer) developed in a previous study, 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from the S2 subunit, and 139 N-glycopeptides from ACE2 were characterized. In addition, we report post-translational modifications (PTMs) of glycan, including mannose-6-phosphate (M6P) and GlcNAc-1-phosphate-6-O-mannose in N-glycan of RBD and ACE2, and O-acetylation in O-glycan of RBD, identified for the first time in these recombinant proteins. The relative abundance distribution according to glycosites and glycan types were analyzed by quantified site-specific N- and O (only from RBD)-glycopeptides from RBD, S2, and ACE2 using I-GPA. Asn331 for RBD, Asn1098 for S2, and Asn103 for ACE2 were majorly N-glycosylated, and dominant glycan-type was complex from RBD and ACE2 and high-mannose from S2. These findings will provide valuable insights into the glycosylation patterns that influence protein function and immunogenicity and offer new perspectives for the development of vaccines and antibody-based therapies against COVID-19. Full article

The tick-borne encephalitis virus is a pathogen endemic to northern Europe and Asia, transmitted through bites from infected ticks. It is a member of the Flaviviridae family and possesses a positive-sense, single-stranded RNA genome encoding a polypeptide that is processed into seven non-structural and three structural proteins, including the envelope (E) protein. The glycosylation of the E protein, involving a single N-linked glycan at position N154, plays a critical role in viral infectivity and pathogenesis. Here, we dissected the entire glycosylation profile of the E protein using liquid chromatography-tandem mass spectrometry and identified three novel O-linked glycans, which were found at relatively low frequency. One of the O-linked glycans was positioned close to the highly conserved N-linked glycan site, and structural analysis suggested that it may be relevant for the function of the E 150-loop. The N154 site was found to be glycosylated with a high frequency, containing oligomannose or complex-type structures, some of which were fucosylated. An unusually high portion of oligomannose N-linked glycan structures exhibited compositions that are normally observed on proteins when they are translocated from the endoplasmic reticulum to the trans-Golgi network, suggesting disruption of the glycan processing pathway in the infected cells from which the E protein was obtained. Full article

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography–tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development. Full article

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC₅₀ of 8.02 nM by binding to glycosylated viral hemagglutinin (K_D of 1.21 × 10⁻⁶ M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans. Full article

The world-wide COVID-19 pandemic has promoted a series of alternative vaccination strategies aiming to elicit neutralizing adaptive immunity in the human host. However, restricted efficacies of these vaccines targeting epitopes on the spike (S) protein that is involved in primary viral entry were observed and putatively assigned to viral glycosylation as an effective escape mechanism. Besides the well-recognized N-glycan shield covering SARS-CoV-2 spike (S) proteins, immunization strategies may be hampered by heavy O-glycosylation and variable O-glycosites fluctuating depending on the organ sites of primary infection and those involved in immunization. A further complication associated with viral glycosylation arises from the development of autoimmune antibodies to self-carbohydrates, including O-linked blood group antigens, as structural parts of viral proteins. This outline already emphasizes the importance of viral glycosylation in general and, in particular, highlights the impact of the site-specific O-glycosylation of virions, since this modification is independent of sequons and varies strongly in dependence on cell-specific repertoires of peptidyl-N-acetylgalactosaminyltransferases with their varying site preferences and of glycan core-specific glycosyltransferases. This review summarizes the current knowledge on the viral O-glycosylation of the SARS-CoV-2 spike protein and its impact on virulence and immune modulation in the host. Full article

Hepatitis viral infections are the most common cause of hepatitis liver disease, which eventually leads to cancer and fibrosis if not detected early. Therefore, early detection would allow for preventive and therapeutic actions. Here, a surface-enhanced Raman spectroscopy (SERS)-based biosensor was developed using plasmonic molybdenum trioxide quantum dots (MoO₃-QDs) as the SERS substrates. The nanostructured substrate of MoO₃-QDs was functionalized with a proteoglycan (syndecan-1) as a novel bioreceptor for the target hepatitis E virus (HEV). The innovative biodetection system achieved a detection limit of 1.05 fg/mL for the tested HEV target (ORF2), indicating superb clinically relevant sensitivity and performance. The designed biosensing system incorporating a glycan motif as a bioreceptor instead of the conventional antibodies or aptamers presents new insights for the ultrasensitive detection of HEV and other infectious viruses. Full article

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, infectivity, and transmissibility. The glycans can mask key epitopes on the S1 protein and alter its structural conformation, allowing the virus to escape the immune system. Therefore, we comprehensively characterize O-glycosylation in eleven variants of SARS-CoV-2 S1 subunits to understand the differences observed in the biology of the variants. In-depth characterization was performed with a double digestion strategy and an efficient LC-MS/MS approach. We observed that O-glycosylation is highly conserved across all variants in the region between the NTD and RBD, whereas other domains and regions exhibit variation in O-glycosylation. Notably, omicron has the highest number of O-glycosylation sites on the S1 subunit. Also, omicron has the highest level of sialylation in the RBD and RBM functional motifs. Our findings may shed light on how differences in O-glycosylation impact viral pathogenicity in variants of SARS-CoV-2 and facilitate the development of a robust vaccine with high protective efficacy against the variants of concern. Full article

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles. Full article

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses. Full article

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells. Full article

In humans and other mammals, the respiratory tract is represented by a complex network of polarized epithelial cells, forming an apical surface facing the external environment and a basal surface attached to the basement layer. These cells are characterized by differential expression of proteins and glycans, which serve as receptors during influenza virus infection. Attachment between these host receptors and the viral surface glycoprotein hemagglutinin (HA) initiates the influenza virus life cycle. However, the virus receptor binding specificities may not be static. Sialylated N-glycans are the most well-characterized receptors but are not essential for the entry of influenza viruses, and other molecules, such as O-glycans and non-sialylated glycans, may be involved in virus-cell attachment. Furthermore, correct cell polarity and directional trafficking of molecules are essential for the orderly development of the system and affect successful influenza infection; on the other hand, influenza infection can also change cell polarity. Here we review recent advances in our understanding of influenza virus infection in the respiratory tract of humans and other mammals, particularly the attachment between the virus and the surface of the polar cells and the polarity variation of these cells due to virus infection. Full article

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells. Full article

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography–mass spectrometry (LC–MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell–cell interactions. Full article