Complex-type N-glycans Research Articles

Effect of N-glycosylation on constitutive signal transduction by mutated cytokine receptor-like factor 2

BackgroundCytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. MethodsHEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. ResultsThree major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. ConclusionsWe found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. General significanceOur studies provide a crucial role of glycosylation in the cell surface expression of receptors.

Cell-free N-glycosylation of peptides using synthetic lipid-linked hybrid and complex N-glycans.

Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate "empty," i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to in vitro N-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human β-1,2N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and β-1,4-galactosyltransferase (GalTΔTM) have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.

Protein N-glycosylation in the bronchoalveolar space differs between never-smokers and long-term smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n=5) and long-term smokers (LTS) with (LTS+, n=4) and without COPD (LTS-, n=8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

Chemoenzymatic synthesis of genetically-encoded multivalent liquid N-glycan arrays

Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N-glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50–1000 copies of dibenzocyclooctyne. The resulting intermediate can be trimmed by glycosidases and extended by glycosyltransferases yielding a phage library with different N-glycans. Post-reaction analysis by MALDI-TOF MS allows rigorous characterization of N-glycan structure and mean density, which are both encoded in the phage DNA. Use of this LiGA with fifteen glycan-binding proteins, including CD22 or DC-SIGN on cells, reveals optimal structure/density combinations for recognition. Injection of the LiGA into mice identifies glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides a quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.

N-glycosylation in non-invasive and invasive intraductal papillary mucinous neoplasm

Intraductal papillary mucinous neoplasms (IPMNs), often found incidentally, are potentially malignant cystic tumors of the pancreas. Due to the precancerous nature, IPMNs lacking malignant features should be kept on surveillance. The follow-up relies on magnetic resonance imaging, which has a limited accuracy to define the high-risk patients. New diagnostic methods are thus needed to recognize IPMNs with malignant potential. Here, aberrantly expressed glycans constitute a promising new area of research. We compared the N-glycan profiles of non-invasive IPMN tissues (n = 10) and invasive IPMN tissues (n = 10) to those of non-neoplastic pancreatic controls (n = 5) by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Both IPMN subgroups showed increased abundance of neutral composition H4N4 and decrease in H3N5F1, increase in sialylation, and decrease in sulfation, as compared to the controls. Furthermore, invasive IPMN showed an increase in terminal N-acetylhexosamine containing structure H4N5, and increase in acidic complex-type glycans, but decrease in their complex fucosylation and sulfation, as compared to the controls. In conclusion, the N-glycan profiles differed between healthy pancreatic tissue and non-invasive and invasive IPMNs. The unique glycans expressed in invasive IPMNs may offer interesting new options for diagnostics.

Complex-Type N-Glycans Are Associated with Cartilage Degeneration within Different Loading Sites of the Tibial Plateau for Knee Osteoarthritis Patients.

Abnormal N-glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N-glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients (n = 8). Subsequently, N-glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N-glycans observed by MALDI-MSI, 3 complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, and 1 oligomannose-type N-glycan, (Hex)9(HexNAc)2, were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N-glycans, (Hex)3(HexNAc)6(Fuc)2 and (Hex)3(HexNAc)6(Fuc)3, were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N-glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.

Efficient production and characterization of soluble active human β-1,2-N-acetylglucosaminyltransferase II in bacteria

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human β-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4μM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E.coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.

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.

Anti-schistosomal immunity to core xylose/fucose in N-glycans.

Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease.

A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-β-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions.

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

N-glycosylation on Asn-57 is required for the correct HAI-2 protein folding and protease inhibitory activity.

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal.

Elucidation of N-/O-glycosylation and site-specific mapping of sialic acid linkage isomers of SARS-CoV-2 human receptor angiotensin-converting enzyme 2.

Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2-3)Gal (over 80.3%) than that of other N-glycosites (35.6-71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells.

Study on antibody Fc-glycosylation for optimal effector functions.

A comprehensive structure-activity relationship study on antibody Fc-glycosylation has been performed using the chimeric anti-SSEA4 antibody chMC813-70 as a model. The α-2,6 sialylated biantennary complex type glycan was identified as the optimal Fc-glycan with significant enhancement in antibody effector functions, including binding to different Fc receptors and ADCC.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 μg ml−1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.