Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concern

Abstract The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation

High-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues are unique to porcine islets.

ObjectivesIslet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection.MethodsN- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS).ResultsA total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets.ConclusionsThese results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors

Limited N-Glycan Processing Impacts Chaperone Expression Patterns, Cell Growth and Cell Invasiveness in Neuroblastoma

Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6–9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis

To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MS^<i>n</i> system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by β elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously

Species-Specific Recognition of Sulfolobales Mediated by UV-Inducible Pili and S-Layer Glycosylation Patterns

Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of Sulfolobales (Ups) pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different Sulfolobus species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species have unique surface-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination

Carbohydrate–Neuroactive Hybrid Strategy for Metabolic Glycan Engineering of the Central Nervous System <i>in Vivo</i>

Sialic acids are abundant in the central nervous system (CNS) and are essential for brain development, learning, and memory. Dysregulation in biosynthesis of sialo-glycoconjugates is known to be associated with neurological disorders, CNS injury, and brain cancer. Metabolic glycan engineering (MGE) and bioorthogonal ligation have enabled study of biological roles of glycans <i>in vivo</i>; however, direct investigations of sialoglycans in brain have been intractable. We report a simple strategy utilizing carbohydrate–neuroactive hybrid (CNH) molecules, which exploit carrier-mediated transport systems available at the blood–brain barrier, to access brain via tail vein injection in mice. Peracetylated <i>N</i>-azidoacetyl-d-mannosamine (Ac₄ManNAz) conjugated with neuroactive carriers, namely, nicotinic acid, valproic acid, theophylline-7-acetic acid, and choline, were synthesized and evaluated in SH-SY5Y (human neuroblastoma) cells for MGE. Intravenous administration of CNH molecules in mice (C57BL/6J and BALB/cByJ) resulted in robust expression of <i>N</i>-azidoacetyl-neuraminic acid (NeuAz)-carrying glycoproteins in both brain and heart, while the nonhybrid molecule Ac₄ManNAz showed NeuAz expression in heart but not in brain. Successful neuroactive carriers were then conjugated with <i>N</i>-butanoyl-d-mannosamine (ManNBut) with a goal to achieve modulation of polysialic acid (polySia) on neural cell adhesion molecules (NCAM). PolySia levels on NCAM in adult mice were reduced significantly upon administration of Ac₃ManNBut-nicotinate hybrid, but not with Ac₄ManNBut. This novel application of MGE not only offers a noninvasive tool for investigating brain glycosylation, which could be developed in to brain mapping applications, but also serves as a potential drug by which modulation of neural glycan biosynthesis and thus function can be achieved <i>in vivo</i>

Inhibition of Mucin-Type <i>O</i>‑Glycosylation through Metabolic Processing and Incorporation of <i>N</i>‑Thioglycolyl‑d‑galactosamine Peracetate (Ac₅GalNTGc)

Mucin-type <i>O</i>-glycans form one of the most abundant and complex post-translational modifications (PTM) on cell surface proteins that govern adhesion, migration, and trafficking of hematopoietic cells. Development of targeted approaches to probe functions of <i>O</i>-glycans is at an early stage. Among several approaches, small molecules with unique chemical functional groups that could modulate glycan biosynthesis form a critical tool. Herein, we show that metabolism of peracetyl <i>N</i>-acyl-d-galactosamine derivatives carrying an <i>N</i>-thioglycolyl (Ac₅GalNTGc, <b>1</b>) moietybut not <i>N</i>-glycolyl (Ac₅GalNGc, <b>2</b>) and <i>N</i>-acetyl (Ac₄GalNAc, <b>3</b>)through the <i>N</i>-acetyl-d-galactosamine (GalNAc) salvage pathway induced abrogation of MAL-II and PNA epitopes in Jurkat cells. Mass spectrometry of permethylated <i>O</i>-glycans from Jurkat cells confirmed the presence of significant amounts of elaborated <i>O</i>-glycans (sialyl-T and disialyl-T) which were inhibited upon treatment with <b>1</b>. <i>O</i>-Glycosylation of CD43, a cell surface antigen rich in <i>O</i>-glycans, was drastically reduced by <b>1</b> in a thiol-dependent manner. By contrast, only mild effects were observed for CD45 glycoforms. Direct metabolic incorporation of <b>1</b> was confirmed by thiol-selective Michael addition reaction of immunoprecipitated CD43-myc/FLAG. Mechanistically, CD43 glycoforms were unperturbed by peracetylated <i>N</i>-(3-acetylthiopropanoyl) (<b>4</b>), <i>N</i>-(4-acetylthiobutanoyl) (<b>5</b>), and <i>N</i>-methylthioacetyl (<b>6</b>) galactosamine derivatives, <i>N</i>-thioglycolyl-d-glucosamine (<b>7</b>, C-4 epimer of <b>1</b>), and α-<i>O</i>-benzyl 2-acetamido-2-deoxy-d-galactopyranoside (<b>8</b>), confirming the critical requirement of both free sulfhydryl and galactosamine moieties for inhibition of mucin-type <i>O</i>-glycans. Similar, yet differential, effects of <b>1</b> were observed for CD43 glycoforms in multiple hematopoietic cells. Development of small molecules that could alter glycan patterns in an antigen-selective and cell-type selective manner might provide avenues for understanding biological functions of glycans

Antigen peptide transporter 1 is involved in the development of fructose-induced hepatic steatosis in mice

Background and Aim: The purpose of this study is to assess whether the decrease in CD8 cells has any role in the development of non-alcoholic fatty liver disease (NAFLD). In this study, we therefore used antigen peptide transporter 1 (TAP1−/−) mice that cannot transport major histocompatibility complex class I antigens onto the cell surface resulting in failure of the generation of CD8 cells. Methods: Wild-type C57Bl/6J and TAP1−/− mice were fed with 30% fructose solution for 8 weeks. The percentage of CD4, CD8 cells in peripheral blood mononuclear cells, and liver were sorted by fluorescence-activated cell sorting in both control and fructose-treated mice. Bodyweight, histopathological changes, oil red O staining, glucose tolerance test, intraperitoneal insulin tolerance test, serum levels of triglycerides, cholesterol, aspartate aminotransferase, and alanine aminotransferase were also evaluated. Quantitative real-time polymerase chain reaction was performed to determine the expression of specific genes involved in development of fatty changes in the liver. Results: Chronic consumption of fructose in TAP1−/− mice did not develop NAFLD, insulin resistance, or change in level of CD8 cells. Moreover, there was delay in relative expression levels of genes involved in development of NAFLD in fructose-treated TAP1−/− mice. Conclusion: Taken together, the data suggest that TAP1−/−-deficient mice displayed reduced levels of CD8 cells that have a vital role in the initiation and propagation of liver inflammation and is a casual role in the beginning of fructose-induced liver damage as well as insulin resistance in mice

Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery