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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 mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-Gsα(KO) 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 myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsα(cont) and Ocy454-Gsα(KO)) 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 Gsα(cont) and Gsα(KO)) 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 Gsα(cont) and Gsα(KO) cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-Gsα(KO) cells compared to Ocy454-Gsα(cont), whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsα(cont) cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-Gsα(KO) cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signalling.

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, β1,6 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 β1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8eligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low β1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed β1,6 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.

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.

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/β-catenin signaling pathway, the β-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that β-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 β-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 β-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.

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., In human endothelial cells, CD209L acts as a receptor for SARS-CoV-2; together with ACE2, it can function as a co-receptor. Blocking CD209L activity inhibited virus entry, indicating a novel target for development of antiviral drugs.

Head and neck cancer is the seventh most common cancer in the world, claiming ~500,000 deaths yearly. Low expression of FUT2, an α1,2-fucosyltransferase that modifies glycoproteins and glycolipids, is associated with lower overall survival in head and neck squamous cell carcinoma (HNSCC). We previously showed that inhibition of nuclear β-catenin/CBP led to an increase in antennary fucosylation of the epidermal growth factor receptor (EGFR) and reduced tumor growth in a mouse orthotopic tumor xenograft model of HNSCC (Chandler KB et al., 2020). One possible explanation for this finding could be attenuation of HNSCC cellular proliferation and survival via restoration of fucose modification-mediated regulation of cell signaling and adhesion, leading to reinforcement of epithelial cell characteristics. To explore this hypothesis, FUT2 was overexpressed in the metastatic HNSCC cell line HSC-3. To compare the rate of proliferation between empty vector (HSC-3-EV) and FUT2-overexpressing cells (HSC-3-FT2), cells were plated in triplicate and photographed every 24 hours using an EVOS Imaging System. Independently, indolent CAL27 cells with high endogenous FUT2 expression and HSC-3-FT2 cells were subject to flow cytometry analyses to probe for fucosylated epitopes on the cell surface. To identify proteins bearing fucosylated determinants, CAL27 and HSC-3 cell lysates were subjected to proteolysis and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS/MS data were searched with PEAKS software (Bioinformatics Solutions, Inc.), and with Byonic (Protein Metrics). Forced expression of FUT2 in HSC-3 cells (HSC-3-FT2) led to a decrease in proliferation compared to HSC-3-EV cells. Flow cytometry analyses revealed the presence of α1,2-fucosylated glycan epitopes on the cell surface of CAL27 cells, but not on parental HSC-3 or HSC-3-EV cells. To probe the role of FUT2 in HNSCC, we sought to identify fucosylated proteins in HNSCC cell lines by applying proteomic and glycoproteomic analyses of CAL27 and HSC-3 cell lysates to identify glycoproteins with differential display of fucosylated epitopes. Among the 1379 proteins in CAL27 cells and 1340 proteins in parental HSC-3 cells (1% FDR cutoff), with 1137 proteins common to both cell lines, we detected at least one glycopeptide for 55 proteins. Adhesion molecules and cell surface receptors were over-represented in the set and will serve as the focus of future inquiry into the mechanism of FUT2-mediated survival in HNSCC. In conclusion, FUT2 overexpression decreases cell proliferation in HSC-3 tongue squamous carcinoma cells. Low endogenous levels of α1,2-fucosylated epitopes were detected on HSC-3 cells with demonstrated metastatic potential, while higher levels were detected on non-metastatic CAL27 cells that express FUT2. We plan to further investigate the role of FUT2 in HNSCC, by exploring the impact of α1,2-fucose modification on signaling and adhesion molecules. Citation Format: Evan Ales, Bach-Cuc Nguyen, Winston H. Elliott, Maria A. Kukuruzinska, Catherine E. Costello, Robert Sackstein, Kevin Brown Chandler. Understanding the role of α1,2-fucosylation in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2004.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy in the world with oral squamous cell carcinomas (OSCC) accounting for the majority of HNSCC cases. A major driver of OSCC is the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) with 12 N-glycosylation sites. Fucosylated N-linked glycans on EGFR are associated with survival e.g., they suppress receptor dimerization and signaling. High levels of fucosylated glycan epitopes have been observed in OSCC, where invasive regions lose expression of linkage-specific fucosylated epitopes, suggesting that fucosylated glycans are involved in the suppression of cell growth and invasion. Previously, it was shown that inhibition of the interaction between nuclear β-catenin and CREB-binding protein (CBP) in human OSCC cells and in mouse tumor xenografts with a small molecule inhibitor ICG-001, interfered with OSCC proliferation and aggressive features in cellular, zebrafish, and murine models. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001 and exhibits ~50 - 100-fold lower EC50 values. Treatment with ICG-001 and E7386, increased expression of two glycosyltransferases, FUT2 and FUT3 coincident with decreased EGFR abundance that was accompanied by higher fucosylation of EGFR and upregulated expression of E-cadherin and junctional β-catenin. Further, genomic analyses showed a positive correlation between the ICG-001 and E7386 treatments and EGFR inhibition, suggesting that higher expression of antennary fucosyltransferase genes suppresses EGFR signaling. We now show using nLC-MS/MS analyses that EGFR from metastatic HSC-3 cells had low levels of fucosylated N-glycans, while EGFR from indolent CAL27 cells displayed higher levels of fucosylation at multiple EGFR N-linked glycosylation sites, and that these changes were statistically significant. In-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry of EGFR glycopeptides revealed new insights into the identity of fucosylated glycan epitopes. Collectively, these results suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of fucosyltransferase expression and activity. We conclude that inhibition of β-catenin/CBP signaling with a novel small molecule E7386 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in HNSCC patients. Citation Format: Kevin B. Chandler, Khalid Alamoud, Bac-Cuc Nguyen, Vanessa L. Stahl, Takashi Owa, Kenichi Nomoto, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of β-catenin/CBP signaling alters EGFR fucosylation status in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5940.

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 H(2)(18)O 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 Hex(3)HexNAc(4)dHex(4) and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex(3)HexNAc(2)dHex(2), each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

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.

Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .

Glycoproteins are well represented among biomarkers for inflammatory and cancer diseases. Secreted and membrane-associated glycoproteins make excellent targets for noninvasive detection. In this review, we discuss clinically applicable markers of cancer diseases and methods for their analysis. High throughput discovery continues to supply marker candidates with unusual glycan structures, altered glycoprotein abundance, or distribution of site-specific glycoforms. Improved analytical methods are needed to unlock the potential of these discoveries in validated clinical assays. A new generation of targeted quantitative assays is expected to advance the use of glycoproteins in early detection of diseases, molecular disease classification, and monitoring of therapeutic interventions.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2, via enzymatic release of the glycans and concomitant incorporation of 18O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for therapeutic targeting of VEGFR-2, ligand binding, trafficking and signaling.

Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed. Harvard University CFAR grant (P30 AI060354); the William F. Milton Fund; the Gilead Sciences Research Scholars Program in HIV; and an Broad-Ragon ENDHIV Catalytic Grant (jointly awarded to D.L. and S.A.C.). C.E.C and K.B.C. were supported by P41 GM104602 and S10 OD010724 grants.

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial receptor tyrosine kinase (RTK), is essential for tumor angiogenesis in tumors of diverse origin. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites. Changes in glycosylation influenced by hypoxia, the shunting of glycolytic intermediates via the hexosamine biosynthesis pathway into glycan synthesis, and pro-inflammatory cytokines have the potential to alter VEGFR-2 signaling in tumors. However, the role of glycosylation in VEGFR-2 signaling and function remain largely unknown. The objective of this study is to determine the functional importance of N-glycosylation in VEGFR-2 activation and angiogenic signaling. Porcine aortic endothelial (PAE) cells with ectopic expression of either wild type VEGFR-2, or one in a series of VEGFR-2 constructs with single glycosylation site mutations in the ligand binding domain, were treated with VEGF-A for 0, 5, 10 or 30 minutes. VEGFR-2 tyrosine phosphorylation was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody to assess activation, and with anti-pTyr-1175-VEGFR-2 and anti-pTyr-1212-VEGFR-2 antibodies. Activation of key molecules involved in downstream signaling of VEGFR-2 was also monitored. To establish the glycosylation status of VEGFR-2 available for ligand-mediated signaling, we isolated plasma membrane localized VEGFR-2, performed enzymatic proteolysis, enriched glycopeptides via hydrophilic interaction liquid chromatography (HILIC), and analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation. Mutation of one N-glycosylation site near the VEGF binding site increased ligand-dependent phosphorylation (at Tyr-1054, Tyr-1175 and Tyr-1212) within 5 min of ligand addition, and initial observations suggest that this resulted in higher levels of activated c-Src compared to wild type VEGFR-2, which may have implications for endothelial cell proliferation and migration. However, phosphorylation of other downstream signaling targets including Akt and PLCγ1 was not affected. Mutations at other N-glycosylation sites near the VEGF ligand binding site did not appear to impact ligand-mediated activation of VEGFR-2, suggesting that glycans located at certain N-linked sites may influence ligand-mediated activation while others have minimal impact. Targeting VEGFR-2 N-glycosylation may be a valuable strategy to inhibit tumor angiogenesis. This research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157. Novel aspect: These results demonstrate that site-specific N-glycosylation of VEGFR-2 modulates ligand-mediated phosphorylation of key VEGFR-2 tyrosine residues involved in downstream signaling. Citation Format: Kevin B. Chandler, Deborah R. Leon, Jenevieve Kuang, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. N-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2047.

Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in >90% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-linked glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/ Β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear Β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small-molecule inhibitor of Β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affected EGFR N-glycosylation. We immunoprecipitated EGFR from indolent CAL27 and metastatic HSC-3 cells after treatment with ICG-001 or vehicle control and determined the effect of inhibition of Β-catenin/CBP activity on its N-glycosylation status. We subjected immunoprecipitated EGFR to proteolysis, performed glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC), analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation, and compared site-specific glycoform patterns for the two cell types +/- ICG-001. At specific N-glycosylation sites, EGFR from indolent CAL27 cells had highly fucosylated N-glycans, while EGFR from metastatic HSC-3 cells displayed N-linked glycans with a paucity of fucose. Treatment of HSC-3 cells with ICG-001 revealed higher fucosylation at sites N151, N420, suggesting that ICG-001 promoted modification with terminal fucose, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3 that fucosylate residues on the outer arms of N-linked glycans. Our studies suggest that the Β-catenin/CBP axis promotes EGFR signaling by inhibiting its fucosylation through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of Β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR protumorigenic activity in OSCC. Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK). Citation Format: Kevin B. Chandler, Khalid Alamoud, Vinay K. Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of Β-catenin/CBP signaling in oral cancer alters EGFR N-glycosylation and abundance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2516.

A series of cyclic lactam analogues of γ-MSH (H-Tyr1-Val2-Met3-Gly4-His5-Phe6-Arg7-Trp8-Asp9-Arg10-Phe11-Gly12-OH) with a bulky hydrophobic residue in the direct proximity to the pharmacophore (Xaa-d-Phe/d-Nal(2′)-Arg-Trp) were designed and synthesized by solid-phase methods. A variety of amino acids with a broad range of hydrophobic/hydrophilic properties was introduced in position 5 to further explore their complementary role in receptor selectivity. Biological evaluation of these peptides revealed several analogues with potent hMC3R agonist and hMC3R/hMC5R antagonist activities, and good receptor selectivity. Analogue 4, c[Nle-Arg-d-Phe-Arg-Trp-Glu]-NH2, was found to be a very potent and selective hMC3R agonist (EC50 = 1.2 nM, 112% act). In addition, analogue 13, c[Nle-Val-d-Nal(2′)-Arg-Trp-Glu]-NH2, was identified as an hMC3R/hMC5R antagonist with the best selectivity against the hMC4R in this series (pA2(hMC3R) = 8.4; pA2(hMC5R) = 8.7). These results indicate the significance of steric factors in melanocortin receptor selectivity and suggest that introduction of bulky residues in the direct proximity to the melanocortin pharmacophore is an effective approach to design of novel hMC3R and hMC5R selective ligands.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR-2 is essential for tumor angiogenesis. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like (Ig) domains, each with multiple potential N-glycosylation sites. N-glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the occupancy and glycoform distributions at each of the potential N-glycosylation sites and their putative role(s) in VEGFR-2 function remain largely unknown. The objective of this study is to investigate the functional importance of VEGFR-2 N-glycosylation in VEGFR-2 angiogenic signaling. Porcine aortic endothelial (PAE) cells with ectopic expression of VEGFR-2 were treated with PNGase F to remove N-linked glycans or heat-denatured PNGase F as a control. Following PNGase F treatment for 4 hours, cells were treated with VEGF-A ligand for 0, 5, 10 or 30 minutes. VEGFR-2 phosphorylation (activation) was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody. In addition, a polyclonal anti-VEGFR2 antibody was used to immunoprecipitate untreated and PNGase F-treated VEGFR-2 from PAE cell lysates. Evaluation of the N-glycosylation sites targeted by PNGase F was carried out by gel electrophoresis, followed by protease digestion and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site-specific glycosylation information we performed proteolysis of VEGFR-2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation. We detected a dramatic increase in ligand-mediated activation of VEGFR-2 after treatment with PNGase F, suggesting that certain N-linked glycans may hinder ligand access to the VEGF binding site, or that removal of N-linked glycans results in conformational changes that lead to increased activation of the receptor by VEGF-A. To explore this observation in greater detail, we have created a series of VEGFR-2 N-glycosylation site mutants, and we are now using the mutants to determine which glycosylation sites are involved in the observed modulation of VEGFR-2 signaling. Citation Format: Kevin B. Chandler, Deborah R. Leon, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. Vascular endothelial growth factor receptor-2 (VEGFR-2) N-glycosylation modulates angiogenic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1808. doi:10.1158/1538-7445.AM2017-1808

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of (18)O water. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction.

Glycoproteins fulfill many indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. Improved analytical methods are needed to allow a complete characterization of this complex and common post-translational modification. In this study, we present a workflow for the analysis of the microheterogeneity of N-glycoproteins that couples hydrophilic interaction and nanoreverse-phase C18 chromatography to tandem QTOF mass spectrometric analysis. A glycan database search program, GlycoPeptideSearch, was developed to match N-glycopeptide MS/MS spectra with the glycopeptides comprised of a glycan drawn from the GlycomeDB glycan structure database and a peptide from a user-specified set of potentially glycosylated peptides. Application of the workflow to human haptoglobin and hemopexin, two microheterogeneous N-glycoproteins, identified a total of 57 distinct site-specific glycoforms in the case of haptoglobin and 14 site-specific glycoforms of hemopexin. Using glycan oxonium ions and peptide-characteristic glycopeptide fragment ions and by collapsing topologically redundant glycans, the search software was able to make unique N-glycopeptide assignments for 51% of assigned spectra, with the remaining assignments primarily representing isobaric topological rearrangements. The optimized workflow, coupled with GlycoPeptideSearch, is expected to make high-throughput semiautomated glycopeptide identification feasible for a wide range of users.

While variable production of the biosurfactant, rhamnolipid, by Pseudomonas aeruginosa has been shown to be dependent on growth conditions, no research has evaluated potential relationships between rhamnolipid production and the presence of heavy metals. The current investigation evaluates the influence of Cd(2+) on rhamnolipid synthesis. Cultures grown in the presence of 0.45 and 0.89 mM Cd(2+) were monitored for rhlB/rhlC expression, rhamnolipid yield, and the ratio of monorhamnolipid (RL1) and dirhamnolipid (RL2) produced. Results show a Cd-induced enhancement of rhlB expression in mid-stationary phase (53 h). In addition, sustained production of rhamnolipid through late stationary growth phase (96 h) was observed for Cd-amended cultures, unlike Cd-free control cultures that ceased rhamnolipid production by mid-stationary growth phase. Most significant was an observed increase in the ratio of RL2 to RL1 congeners produced by cultures grown in the presence of Cd(2+). Previous results have shown that the complexation constant for RL2-Cd is several orders of magnitude larger than that of RL1-Cd thus the preferential production of RL2 in the presence of Cd(2+) impacts its bioavailability and toxicity both for the cell and in the surrounding environment.

Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9(PG-20N) is a selective and competitive hMC5R antagonist, with IC 50 of 130 +/- 11 nM, and a pA 2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC 50 values of 38 +/- 3 nM and 58 +/- 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH(2)) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH(2)) by replacing the His-d-Phe and His-d-Nal(2') fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx=D-Phe/pCl-D-Phe/D-Nal(2')). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.

A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.

Introduction The natural melanotropin peptides, which include -, -, -melanocyte-stimulating hormone (MSH), and adrenocorticotropin (ACTH), are derived by posttranslational processing of the pro-opiomelanocortin (POMC) gene transcript. Each of them possesses a central “core” sequence His-Phe-Arg-Trp, which is essential for their agonist biological activity. It has been discovered that the melanocortin receptors (hMCR) and their ligands control a surprisingly large number of multifaceted biological actions including skin pigmentation, erectile function, blood pressure and heart rate, control of feeding behavior, and effects on memory and learning processes [1,2]. Of particular interest for pharmaceutical research are the hMC3R and the hMC4R, which have been implicated to play complementary roles in weight control [3]. Therefore, selective ligands for these receptors may provide a novel approach in the treatment of obesity, anorexia, weight loss, and related disorders. Recent reports [4] have shown that some 21-membered or larger cyclic lactam analogs of and -MSH are potent and hMC4R-selective agonists. At the same time, Kavarana et al. [5] have found that enhancing the hydrophobicity of the cyclic peptide combined with the increased ring size resulted in improved hMC3R selectivity. From these observations, we designed a series of novel cyclic -MSH analogs with the following general sequence: c[Nle-Xaa-D-Phe/D-Nal(2’)-Arg-TrpGlu]-NH2. We introduced a bulky hydrophobic residue (Nle ) in the close proximity to the pharmacophore (Xaa-D-Phe/D-Nal(2’)-Arg-Trp) to investigate the impact of steric hindrance on receptor selectivity. Also, a variety of amino acids with a broad range of hydrophobic/hydrophilic properties were placed in position 5 to further explore their complementary role in receptor selectivity (Fig. 1).

Purpose: Incidence of hepatocellular cancer (HCC) continues to increase primarily due to hepatitis C viral (HCV) infection. Prognosis and survival of patients is highly affected by the disease stage at the time of diagnosis. Our mass spectrometric study evaluated N-glycans during the progression of HCV infection to cancer. Glycosylation of immunoglobulins and other serum proteins was examined in cultured cells and in serum of patients with hepatocellular carcinoma. Methods and Results: Protein associated N-glycans were released with PNGaseF and analyzed by MALDI-TOF/TOF analysis following solid phase permethylation. Analysis of less than 0.04 ml of serum led to relative quantification of 70 N-glycan structures. Immunoaffinity isolation of immunoglobulins and other serum proteins allowed us to study protein-specific glycosylation. Progression of HCV infection to HCC was strongly associated with changes in the glycosylation of immunoglobulins. In a pilot case-control study (25 HCC cases and 35 controls), N-glycosylation of eight glycans was significantly different in HCC compared to chronic liver disease controls. The glycosylation of serum proteins was further compared to the proteins secreted by the Huh-7.5 cell line. Conclusion: This study demonstrates mass spectrometric analysis of 70 N-glycans in serum, cultured cells, and isolated proteins. Our results show that the analysis of permethylated N-glycans helps to define changes associated with the progression of HCV infection to HCC. Evaluation of glycan abundance suggests the potential to use glycans for the detection of liver disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5569.