Gas-Phase Structures of Fucosylated Oligosaccharides: Alkali Metal and Halogen Influences.

Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li+, Na+, K+, Rb+, and Cs+) and halogen anions (Cl-, Br-, and I-) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li]+ molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I]- molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

Clinical glycoprotein mass spectrometry: The future of disease detection and monitoring.

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease.

Site-Specific Glycosylation Analysis of Human and Murine Fcγ Receptor II Family Members Reveals Variant-Specific N-Glycosylation.

Fcγ-receptors (FcγRs) including FcγRII (CD32) gene family members are expressed on leukocytes, bind the crystallizable fragment (Fc) region of immunoglobulin G (IgG), and bridge humoral and cellular immunity. FcγRIIA and FcγRIIB have opposing roles, with the former responsible for activation and the latter for inhibition of immune cell signaling and effector functions. The extracellular domains of human and murine FcγRIIs share multiple conserved N-glycosylation sites. Understanding the role(s) of FcγRIIA and FcγRIIB glycosylation in autoimmune diseases is precluded by a lack of effective methods to study disease-associated changes in glycosylation. To address this barrier, we developed a method to assess site-specific glycosylation of human FcγRIIA and FcγRIIB, and the mouse ortholog of human FcγRIIB. Among the receptors, conserved glycosylation sites are compared, with the N144/145 site displaying predominantly complex glycans in recombinant FcγRIIs. Differences in sialylation between recombinant human FcγRIIA H/R134 (H/R131) variants at a nearby N145 N-glycosylation site are reported. Further, a potential human FcγRIIA O-glycosylation site, S179 (S212), is reported in recombinant FcγRIIA. The robust method to assess site-specific glycosylation of FcγRIIs reported here, can be utilized to study the potential role of FcγRII family glycosylation in disease. Data are available via ProteomeXchange with identifier PXD049429.

Cross-platform proteomics signatures of extreme old age.

In previous work, we used a SomaLogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood generated in an independent set. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals. The comparison with blood transcriptomics also highlights a possible role for neutrophil degranulation in aging.

Cross-platform proteomics signatures of extreme old age.

In previous work we used a Somalogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals.

Site-Specific Glycosylation Analysis of Murine and Human Fcγ Receptors Reveals High Heterogeneity at Conserved N-Glycosylation Site.

Fc γ-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions. Dysregulation of FcγR-mediated processes contributes to multiple inflammatory diseases, including rheumatoid arthritis, lupus, and immune thrombocytopenia. Critically, immunoregulatory N-glycan modifications on both FcγRs and IgGs alter FcγR-IgG binding affinity. Rapid methods for the characterization of N-glycans across multiple Fcγ receptors are needed to propel investigations into disease-specific contributions of FcγR N-glycans. Here, we utilize nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) to characterize FcγR glycosylation and report quantitative and site-specific N-glycan characterization of recombinant human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 from CHO cells and murine FcγRI, FcγRIII, and FcγRIV from NS0 cells. Data are available via ProteomeXchange with identifier PXD043966. Broad glycoform distribution (≥30) was observed at mouse FcγRIV site N159 and human FcγRIIIA site N162, an evolutionarily conserved site. Further, mouse FcγRIII N-glycopeptides spanning all four predicted N-glycosylation sequons were detected. Glycoform relative abundances for hFcγRIIIA V/F158 polymorphic variants are reported, demonstrating the clinical potential of this workflow to measure differences in glycosylation between common human FcγRIIIA allelic variants with disease-associated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.

Enrichment and nLC-MS/MS Analysis of Head and Neck Cancer Mucinome Glycoproteins.

Mucin-domain glycoproteins expressed on cancer cell surfaces play central roles in cell adhesion, cancer progression, stem cell renewal, and immune evasion. Despite abundant evidence that mucin-domain glycoproteins are critical to the pathobiology of head and neck squamous cell carcinoma (HNSCC), our knowledge of the composition of that mucinome is grossly incomplete. Here, we utilized a catalytically inactive point mutant of the enzyme StcE (StcEE447D) to capture mucin-domain glycoproteins in head and neck cancer cell line lysates followed by their characterization using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), and enrichment analyses. We demonstrate the feasibility of this workflow for the study of mucin-domain glycoproteins in HNSCC, identify a set of mucin-domain glycoproteins common to multiple HNSCC cell lines, and report a subset of mucin-domain glycoproteins that are uniquely expressed in HSC-3 cells, a cell line derived from a highly aggressive metastatic tongue squamous cell carcinoma. This effort represents the first attempt to identify mucin-domain glycoproteins in HNSCC in an untargeted, unbiased analysis, paving the way for a more comprehensive characterization of the mucinome components that mediate aggressive tumor cell phenotypes. Data associated with this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029420.

Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry.

Bottom-up nLC-MS/MS-based glycoprotein mass spectrometry workflows rely on the generation of a mixture of non-glycosylated and glycosylated peptides via proteolysis of glycoproteins. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable non-glycosylated peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present a potential benefit for glycoproteomic workflows by enabling orthogonal separation of non-glycosylated peptides and glycopeptides following chromatographic separation, and prior to MS/MS analysis. However, knowledge is lacking regarding optimal FAIMS conditions for glycopeptide analyses. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignment confidence by comparing the number of assigned glycopeptides at different confidence levels using a standard nLC-MS/MS method or an otherwise identical method employing FAIMS. Optimized methods will potentiate glycoproteomic analyses by increasing the number of unique glycopeptide identifications and the confidence of glycopeptide assignments. Data are available via ProteomeXchange with identifier PXD036667. Analysis of alpha-1-acid glycoprotein (AGP) tryptic digests via nLC-FAIMS-MS/MS (top) led to the establishment of ideal FAIMS voltages for the analysis of AGP N-glycopeptides (bottom), suggesting that FAIMS can improve the depth of glycoproteome characterization. Pairs of CV magnitudes are shown along the x-axis.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, ß1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Identification of α1,2-fucosylated signaling and adhesion molecules in head and neck squamous cell carcinoma.

Head and neck cancer is the seventh most common cancer in the world, and most cases manifest as head and neck squamous cell carcinoma. Despite the prominent role of fucosylated carbohydrate antigens in tumor cell adhesion and metastasis, little is known about the functional role of fucose-modified glycoproteins in head and neck cancer pathobiology. Inactivating polymorphisms of the fut2 gene, encoding for the α1,2-fucosyltransferase FUT2, are associated with an increased incidence of head and neck cancer among tobacco users. Moreover, the presence of the α1,2-fucosylated Lewis Y epitope, with both α1,2- and α1,3-linked fucose, has been observed in head and neck cancer tumors while invasive regions lose expression, suggesting a potential role for α1,2-fucosylation in the regulation of aggressive tumor cell characteristics. Here, we report an association between fut2 expression and head and neck cancer survival, document differential surface expression of α1,2-fucosylated epitopes in a panel of normal, dysplastic, and head and neck cancer cell lines, identify a set of potentially α1,2-fucosylated signaling and adhesion molecules including the epidermal growth factor receptor (EGFR), CD44 and integrins via tandem mass spectrometry, and finally, present evidence that EGFR is among the α1,2-fucosylated and LeY-displaying proteins in head and neck cancer. This knowledge will serve as the foundation for future studies to interrogate the role of LeY-modified and α1,2-fucosylated glycoproteins in head and neck cancer pathogenesis. Data are available via ProteomeXchange with identifier PXD029420.

The cell adhesion molecule TMIGD1 binds to moesin and regulates tubulin acetylation and cell migration.

BACKGROUND: The cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell-cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated. RESULTS: TMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of α-tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of α-tubulin and filamentous (F)-actin organization. CONCLUSIONS: TMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of α-tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.

CD209L/L-SIGN and CD209/DC-SIGN Act as Receptors for SARS-CoV-2.

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelia and endothelia. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor-binding domain (S-RBD) or S1 encompassing both N termal domain and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection, and interference with CD209L activity by a knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent and may have implications for antiviral drug development.

Protein signatures of centenarians and their offspring suggest centenarians age slower than other humans.

Using samples from the New England Centenarian Study (NECS), we sought to characterize the serum proteome of 77 centenarians, 82 centenarians' offspring, and 65 age-matched controls of the offspring (mean ages: 105, 80, and 79 years). We identified 1312 proteins that significantly differ between centenarians and their offspring and controls (FDR < 1%), and two different protein signatures that predict longer survival in centenarians and in younger people. By comparing the centenarian signature with 2 independent proteomic studies of aging, we replicated the association of 484 proteins of aging and we identified two serum protein signatures that are specific of extreme old age. The data suggest that centenarians acquire similar aging signatures as seen in younger cohorts that have short survival periods, suggesting that they do not escape normal aging markers, but rather acquire them much later than usual. For example, centenarian signatures are significantly enriched for senescence-associated secretory phenotypes, consistent with those seen with younger aged individuals, and from this finding, we provide a new list of serum proteins that can be used to measure cellular senescence. Protein co-expression network analysis suggests that a small number of biological drivers may regulate aging and extreme longevity, and that changes in gene regulation may be important to reach extreme old age. This centenarian study thus provides additional signatures that can be used to measure aging and provides specific circulating biomarkers of healthy aging and longevity, suggesting potential mechanisms that could help prolong health and support longevity.

CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2.

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.

Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms.

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.

N-glycosylation in Spodoptera frugiperda (Lepidoptera: Noctuidae) midgut membrane-bound glycoproteins.

Spodoptera frugiperda is a widely distributed agricultural pest. It has previously been established that glycoproteins in the midgut microvillar membrane of insects are targets for toxins produced by different organisms as well as plant lectins. However, there is still little information about the N-glycome of membrane-bound midgut glycoproteins in Lepidoptera and other insect groups. The present study used mass spectrometry-based approaches to characterize the N-glycoproteins present in the midgut cell microvilli of Spodoptera frugiperda. We subjected midgut cell microvilli proteins to proteolytic digestion and enriched the resulting glycopeptides prior to analysis. We also performed endoglycosidase release of N-glycans in the presence of H218O determining the compositions of released N-glycans by MALDI-TOF MS analysis and established the occupancy of the potential N-glycosylation sites. We report here a total of 160 glycopeptides, representing 25 N-glycan compositions associated with 70 sites on 35 glycoproteins. Glycan compositions consistent with oligomannose, paucimannose and complex/hybrid N-glycans represent 35, 30 and 35% of the observed glycans, respectively. The two most common N-glycan compositions were the complex/hybrid Hex3HexNAc4dHex4 and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex3HexNAc2dHex2, each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

The role of leached Zr in the photocatalytic reduction of CO2 to formate by derivatives of UiO-66 metal organic frameworks.

This work reports the photocatalytic reduction of CO2 to formate that is mediated by UiO-66 Zr MOF derivatives. Amino-substituted UiO-66 is a known photocatalyst for this transformation, and herein we identify that this catalysis is due to leached Zr, not the MOF itself. No correlation between catalytic activity and crystal size is observed for UiO-66-NH2. Recycling studies along with SEM images of the crystals prior to and after catalysis support our conclusion that the catalytic performance can be described by the amount of leached Zr. Moreover, when the effect of the linker on the catalytic reaction is probed, all MOFs that facilitate the reduction of CO2 are found to leach Zr into solution. Correlation of the MOF (or linker) band gap energies to formate production indicates that this is an important parameter to the leached species. Combined with a linker exchange study, this indicates that the leached Zr still coordinates the linker. These results indicate that the UiO-66 Zr MOFs are not stable under typical photochemical conditions, and emphasize the importance of considering the role that leached metals play in catalysis.

ß-Catenin/CBP inhibition alters epidermal growth factor receptor fucosylation status in oral squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/ß-catenin signaling pathway, the ß-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that ß-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of ß-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that ß-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2).

The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N-glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid-capped N-glycans. VEGFR2 is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. Here, using glycoside hydrolase and kinase assays and immunoprecipitation and MS-based analyses, we demonstrate that N-linked glycans at the Asn-247 site in VEGFR2 hinder VEGF ligand-mediated receptor activation and signaling in endothelial cells. We provide evidence that cell surface-associated VEGFR2 displays sialylated N-glycans at Asn-247 and, in contrast, that the nearby sites Asn-145 and Asn-160 contain lower levels of sialylated N-glycans and higher levels of high-mannose N-glycans, respectively. Furthermore, we report that VEGFR2 Asn-247-linked glycans capped with sialic acid oppose ligand-mediated VEGFR2 activation, whereas the uncapped asialo-glycans favor activation of this receptor. We propose that N-glycosylation, specifically the capping of N-glycans at Asn-247 by sialic acid, tunes ligand-dependent activation and signaling of VEGFR2 in endothelial cells.

Glycosylation in the Tumor Microenvironment: Implications for Tumor Angiogenesis and Metastasis.

Just as oncogene activation and tumor suppressor loss are hallmarks of tumor development, emerging evidence indicates that tumor microenvironment-mediated changes in glycosylation play a crucial functional role in tumor progression and metastasis. Hypoxia and inflammatory events regulate protein glycosylation in tumor cells and associated stromal cells in the tumor microenvironment, which facilitates tumor progression and also modulates a patient's response to anti-cancer therapeutics. In this review, we highlight the impact of altered glycosylation on angiogenic signaling and endothelial cell adhesion, and the critical consequences of these changes in tumor behavior.