N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer. SIGNIFICANCE: This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.

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

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.

Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies.

The tumor-associated antigen mesothelin is expressed at high levels on the cell surface of many human cancers, while its expression in normal tissues is limited. The binding of mesothelin to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. Immunotherapeutic strategies targeting mesothelin are being intensively investigated. Here, we report the crystal structures of mesothelin that reveal a compact, right-handed solenoid consisting of 24 short helices and connecting loops. These helices form a nine-layered spiral coil that resembles ARM/HEAT family proteins. Glycan attachments have been identified in the structure for all three predicted N-glycosylation sites and confirmed with samples from cell culture and patient ascites. The structures of full-length mesothelin and its complex with the Fab of MORAb-009 reveal the interaction of the antibody with the complete epitope, which has not been reported previously. The N-terminal half of mesothelin is conformationally rigid, suitable for eliciting specific antibodies, whereas its C-terminal portion is more flexible. The structure of the C-terminal shedding-resistant fragment of mesothelin complexed with a mAb 15B6 displays an extended linear epitope and helps explain the protection afforded by the antibody for the shedding sites. Significance: The structures of full-length mesothelin and its complexes with antibodies reported here are the first to be determined experimentally, providing atomic models for structural organization of this protein and its interactions with antibodies. It offers insights into the function of mesothelin and guidance for further development of therapeutic antibodies.

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.

Development and validation of a mass spectrometry based analytical method to quantify the ratios in hemagglutinin trimers in quadrivalent influenza nanoparticle vaccine - FluMos-v1.

A quadrivalent influenza nanoparticle vaccine (FluMos-v1) offers long-lasting protection against multiple influenza virus strains and is composed of four strains of hemagglutinin trimer (HAT) assembled around a pentamer core. Here we report an LC-MS/MS analytical development and validation method to measure the percentage of each HAT component in FluMos-v1.

Site Specific N- and O-glycosylation mapping of the Spike Proteins of SARS-CoV-2 Variants of Concern.

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 led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profile 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 (NTD) 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.

Origin of cytoplasmic GDP-fucose determines its contribution to glycosylation reactions.

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules, or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here, we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have a general applicability.

Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin.

SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface.

N-glycans are displayed on cell-surface proteins and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photo-cross-linker onto N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.

Desialylation of O-glycans activates von Willebrand factor by destabilizing its autoinhibitory module.

BACKGROUND: The binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. OBJECTIVES: To understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity. METHODS: A recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. RESULTS: Asialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter. CONCLUSIONS: Desialylation of O-glycans around A1 increases its activity by destabilizing the AIM.

Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure.

Campylobacter jejuni is a significant cause of human gastroenteritis worldwide, and all strains express an N-glycan that is added to at least 80 different proteins. We characterized 98 C. jejuni isolates from infants from 7 low- and middle-income countries and identified 4 isolates unreactive with our N-glycan-specific antiserum that was raised against the C. jejuni heptasaccharide composed of GalNAc-GalNAc-GalNAc(Glc)-GalNAc-GalNAc-diNAcBac. Mass spectrometric analyses indicated these isolates express a hexasaccharide lacking the glucose branch. Although all 4 strains encode the PglI glucosyltransferase (GlcTF), one aspartate in the DXDD motif was missing, an alteration also present in ∼4% of all available PglI sequences. Deleting this residue from an active PglI resulted in a nonfunctional GlcTF when the protein glycosylation system was reconstituted in E. coli, while replacement with Glu/Ala was not deleterious. Molecular modeling proposed a mechanism for how the DXDD residues and the structure/length beyond the motif influence activity. Mouse vaccination with an E. coli strain expressing the full-length heptasaccharide produced N-glycan-specific antibodies and a corresponding reduction in Campylobacter colonization and weight loss following challenge. However, the antibodies did not recognize the hexasaccharide and were unable to opsonize C. jejuni isolates lacking glucose, suggesting this should be considered when designing N-glycan-based vaccines to prevent campylobacteriosis.

Glycosylation of SARS-CoV-2: structural and functional insights.

The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Similar to other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. S, E, and M proteins are glycosylated, and the N protein is phosphorylated. The S protein is involved in the interaction with the host receptor human angiotensin-converting enzyme 2 (hACE2), which is also heavily glycosylated. Recent studies have revealed several other potential host receptors or factors that can increase or modulate the SARS-CoV-2 infection. Interestingly, most of these molecules bear carbohydrate residues. While glycans acquired by the viruses through the hijacking of the host machinery help the viruses in their infectivity, they also play roles in immune evasion or modulation. Glycans play complex roles in viral pathobiology, both on their own and in association with carrier biomolecules, such as proteins or glycosaminoglycans (GAGs). Understanding these roles in detail can help in developing suitable strategies for prevention and therapy of COVID-19. In this review, we sought to emphasize the interplay of SARS-CoV-2 glycosylated proteins and their host receptors in viral attachment, entry, replication, and infection. Moreover, the implications for future therapeutic interventions targeting these glycosylated biomolecules are also discussed in detail.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

Nuclear receptors FXR and SHP regulate protein N-glycan modifications in the liver.

Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are key regulators of metabolism. Here, we report a previously unknown function for the hepatic FXR-SHP axis in controlling protein N-linked glycosylation. Transcriptome analysis in liver-specific Fxr-Shp double knockout (LDKO) livers revealed induction of genes encoding enzymes in the N-glycosylation pathway, including Mgat5, Fut8, St3gal6, and St6gal1 FXR activation suppressed Mgat5, while Shp deletion induced St3gal6 and St6gal1 Increased percentages of core-fucosylated and triantennary glycan moieties were seen in LDKO livers, and proteins with the "hyperglycoforms" preferentially localized to exosomes and lysosomes. This up-regulation of N-glycosylation machinery was specific to the Golgi apparatus and not the endoplasmic reticulum. The increased glycan complexity in the LDKO correlated well with dilated unstacked Golgi ribbons and alterations in the secretion of albumin, cholesterol, and triglycerides. Our findings demonstrate a role for the FXR-SHP axis in maintaining glycoprotein diversity in the liver.

Glycoproteomic Sample Processing, LC-MS, and Data Analysis Using GlycReSoft.

Identification of N- and O-glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro- and micro-heterogeneous and alters the dissociation behavior of glycopeptides, specific sample preparation, mass spectrometry, and data analysis techniques are required. Advanced tandem mass spectrometry-based glycoproteomics coupled with powerful data mining algorithms are key elements for characterization of protein glycosylation. This article includes the detailed, streamlined sample preparation method for liquid chromatography-mass spectrometry data acquisition and subsequent bioinformatics-based data annotation using the publicly available GlycReSoft program for highly efficient identification and quantification of glycoprotein glycosylation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Characterization of glycans and site occupancy on purified glycoprotein Support Protocol 1: In-gel digestion of glycoproteins Support Protocol 2: Detection of glycoproteins from cells/tissue through glycopeptide enrichment Basic Protocol 2: Acquisition of glycopeptides through high-resolution nano-LC-MS/MS Basic Protocol 3: Identification and quantification of glycopeptides using GlycReSoft.

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.

Vaccines and Therapeutics for COVID-19 - How Can Understanding SARS-CoV-2 Glycosylation Lead to Pharmaceutical Advances?

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people worldwide. Researchers have targeted the SARS-CoV-2 structural proteins to better combat the pandemic. Of the four structural proteins, spike (S), membrane (M), envelope (E) and nucleocapsid (N), the S, M and E proteins are glycosylated whereas the N protein is phosphorylated. The glycosylation of the S protein has been reported previously by multiple research groups, and this knowledge has assisted the pharmaceutical industry in developing vaccines and treatment options. In the United States, there are currently three approved COVID-19 vaccines. All three of these vaccines use the S protein to teach host cells how to react when SARS-CoV-2 particles are present. Treatment options utilizing antivirals and immunosuppressants are being developed in addition to vaccines. Different treatment approaches are needed based on the severity of COVID-19 infection. The therapeutic options currently available are not derived through the direct knowledge on SARS-CoV-2 glycosylation. However, more research on the glycosylation of the structural proteins and how this effects SARS-CoV-2 and host cell binding could lead to new and more effective therapeutics. Herein we outline the current vaccine and therapeutic options against COVID-19 available to the public, as well as those still in development.

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

OBJECTIVES: Islet 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. METHODS: N- 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). RESULTS: A 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. CONCLUSIONS: These 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.

Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation.

Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.