The bone morphogenetic protein receptor type 2 (BMPR2) is expressed in multiple cell types, including endothelial cells, and mediates cell signaling via binding of transforming growth factor β (TGF-β) family ligands, contributing to balanced cell proliferation, apoptosis, and angiogenesis. BMPR2 N-glycosylation reportedly influences ligand binding. Here, we sought to develop a bottom-up nanoliquid chromatography-tandem mass spectrometry method to obtain evidence of site-specific N-glycosylation of human BMPR2. To optimize the observation of all three predicted N-glycosylation sites, three protease combinations were tested. Trypsin-Glu-C N-glycopeptides spanning all three putative BMPR2 N-glycosylation sites (N29, N84, and N100) were observed. Analyses of N-glycosylation site occupancy revealed that sites N29 and N84 are highly occupied, while a minor but detectable proportion of BMPR2 is not glycosylated at site N100. Glycopeptide analyses revealed N-glycan compositions consistent with hybrid and complex glycoforms with abundant fucosylation and sialylation. The nLC-MS/MS method established here will facilitate the study of BMPR2 N-glycosylation and its function in homeostasis and disease. Data are available via ProteomeXchange with identifier PXD066494.

The Fc γ-receptor IIIa (FcγRIIIa), or CD16a, is a receptor for the Fc region of IgG that plays an essential role in the regulation of antibody effector functions. Dysregulated CD16a activity contributes to tissue damage and chronic inflammation or, conversely, compromises the removal of immune complexes. CD16a glycoforms with high-mannose N-linked glycans at site N162 demonstrate higher affinity for IgG compared to other forms. Here, we developed a targeted nanoliquid chromatography tandem mass spectrometry parallel reaction monitoring (PRM) method for the site-specific study of CD16a N-glycosylation. The method was applied to assess changes in site N162 N-glycosylation in a model of monocyte differentiation and in CD16a from human primary monocytes and peripheral blood mononuclear cells. Site N162 N-glycosylation was similar across the surveyed cell types. An increase in the proportion of high-mannose N-glycan compositions at site N162 was observed, corresponding to high-affinity glycoforms of CD16a, in a model of monocyte differentiation. Here, the feasibility of using targeted glycopeptide PRM to study CD16a glycosylation in primary cells has been demonstrated. This strategy is well-suited for adaptation to clinical studies focused on the role of CD16a glycosylation in physiology, immunotherapy, and immune disorders. Data are available via ProteomeXchange with the identifier PXD071784.

Pulmonary vascular disease (PVD) encompasses a group of conditions that impact the blood vessels in the lungs. Endothelial cells, which line blood vessels, bear a conspicuous layer of glycosylated molecules that project into the vascular lumen. Recent findings support a role for endothelial cell glycosylation in the development and progression of PVD. However, glycosylation remains a critically understudied aspect of pulmonary vascular health and disease. Here, we provide an overview of the functional role of endothelial cell glycosylation, present evidence of the involvement of glycosylation in PVD, and outline gaps in knowledge regarding the role of glycosylation within the pulmonary vasculature, to provide a foundation for future research efforts focused on advancing glycosylation pathway-targeted treatments for pulmonary vascular disease. Authors review the involvement of glycosylation in pulmonary vascular disease and outline gaps in knowledge to provide a foundation for future efforts focused on advancing glycosylation pathway-targeted treatments.

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.

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.

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.

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.

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.

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.

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, (beta 1,6 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 gly-come-associated genes and found that (beta 1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low (beta 1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed (beta 1,6 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.

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

Purpose/Objective(s) Head and neck cancer is the sixth most common tumor type worldwide, but despite advances in treatment, the 5-year survival rate is only ∼50%. Glycosylation, a common post-translational modification, is frequently altered in cancer. Changes in glycosyltransferase expression in cancer are associated with cellular invasion, tumor growth, and metastasis. However, the role of glycosyltransferase expression in head and neck cancer survival is poorly understood. Here, we examine the association between glycosyltransferase expression and survival in head and neck squamous cell carcinoma (HNSCC). Materials/Methods A retrospective cohort study was performed with data from the Cancer Genome Atlas (TCGA) HNSCC dataset (n=499), containing expression (mRNA) level data, generated via high-throughput sequencing, of primary tumor tissue from patients with a diagnosis of HNSCC (deidentified). A set of glycosyltransferase genes: FUT1-FUT11, POFUT1-2, ST3Gal1-6, ST6Gal1-2, ST6GalNac1-6, ST8Sia1-6, GALNT2-3, C1GALT1, B4GALT4, GCNT2 were considered. Median expression was used to delineate high and low expression groups. Univariate Kaplan-Meier analyses and logrank tests were conducted. Multivariate analyses were performed via Cox proportional hazards regression to adjust for covariates (HPV status, age at initial diagnosis, sex, alcohol history, tobacco usage, and primary site). Results Based on the Kaplan-Meier analysis of FUT2, the median survival time (MST) of the high FUT2-expressing group was 4.7 years, while low expression was associated with an MST of 2.7 years. Cox regression analysis yielded adjusted hazard ratio (aHR) of 0.72 (CI = 0.54-0.95). The MST of the FUT6 high expression group was 4.7 years versus 2.9 years for low expression, with an aHR of 0.62 (CI = 0.47-0.83). The MST of the high-FUT7 expressing group was 4.7 years versus 3.5 years for the low level group, with an aHR of 0.72 (CI = 0.54-0.95). GALNT3 showed the opposite effect, with high expression yielding an MST of 3.1 years versus 4.9 years for the low-expressing group, with aHR of 1.47 (1.11-1.95). An aHR of <1 indicates a protective effect from high expression. Conclusion Overexpression of FUT2, FUT6, and FUT7 were associated with increased overall survival, while overexpression of GALNT3 was associated with decreased overall survival in HNSCC. FUT2 is expressed in endoderm-derived epithelial tissue including the digestive tract and salivary glands, while FUT6 and FUT7 are prominently expressed in immune cells. Thus, FUT6 and FUT7 expression may indicate immune cell infiltration into tumor tissue. Future studies will seek to sort tumor tissue based on cell type, to gain a better understanding of the cell-type specific glycosyltransferase expression in HNSCC tumors. The glycosyltransferase genes associated with survival in this study may serve as future drug targets in head and neck cancer. Head and neck cancer is the sixth most common tumor type worldwide, but despite advances in treatment, the 5-year survival rate is only ∼50%. Glycosylation, a common post-translational modification, is frequently altered in cancer. Changes in glycosyltransferase expression in cancer are associated with cellular invasion, tumor growth, and metastasis. However, the role of glycosyltransferase expression in head and neck cancer survival is poorly understood. Here, we examine the association between glycosyltransferase expression and survival in head and neck squamous cell carcinoma (HNSCC). A retrospective cohort study was performed with data from the Cancer Genome Atlas (TCGA) HNSCC dataset (n=499), containing expression (mRNA) level data, generated via high-throughput sequencing, of primary tumor tissue from patients with a diagnosis of HNSCC (deidentified). A set of glycosyltransferase genes: FUT1-FUT11, POFUT1-2, ST3Gal1-6, ST6Gal1-2, ST6GalNac1-6, ST8Sia1-6, GALNT2-3, C1GALT1, B4GALT4, GCNT2 were considered. Median expression was used to delineate high and low expression groups. Univariate Kaplan-Meier analyses and logrank tests were conducted. Multivariate analyses were performed via Cox proportional hazards regression to adjust for covariates (HPV status, age at initial diagnosis, sex, alcohol history, tobacco usage, and primary site). Based on the Kaplan-Meier analysis of FUT2, the median survival time (MST) of the high FUT2-expressing group was 4.7 years, while low expression was associated with an MST of 2.7 years. Cox regression analysis yielded adjusted hazard ratio (aHR) of 0.72 (CI = 0.54-0.95). The MST of the FUT6 high expression group was 4.7 years versus 2.9 years for low expression, with an aHR of 0.62 (CI = 0.47-0.83). The MST of the high-FUT7 expressing group was 4.7 years versus 3.5 years for the low level group, with an aHR of 0.72 (CI = 0.54-0.95). GALNT3 showed the opposite effect, with high expression yielding an MST of 3.1 years versus 4.9 years for the low-expressing group, with aHR of 1.47 (1.11-1.95). An aHR of <1 indicates a protective effect from high expression. Overexpression of FUT2, FUT6, and FUT7 were associated with increased overall survival, while overexpression of GALNT3 was associated with decreased overall survival in HNSCC. FUT2 is expressed in endoderm-derived epithelial tissue including the digestive tract and salivary glands, while FUT6 and FUT7 are prominently expressed in immune cells. Thus, FUT6 and FUT7 expression may indicate immune cell infiltration into tumor tissue. Future studies will seek to sort tumor tissue based on cell type, to gain a better understanding of the cell-type specific glycosyltransferase expression in HNSCC tumors. The glycosyltransferase genes associated with survival in this study may serve as future drug targets in head and neck cancer.

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.

Introduction: Head and neck (H&N) cancers account for 5-7% of new cancer cases in the United States, and most manifest as head and neck squamous cell carcinomas (HNSCC). Fucosyltransferases (FUTs) contribute to the formation of sialofucosylated epitopes, which alter cellular signaling and mediate early steps in metastasis. However, few studies have examined the effect of FUT expression on H&N cancer survival. Here, we sought to examine the association between fucosyltransferase expression and H&N survival and investigate the mechanistic role of fucosylation in H&N cancer. Methods: FUT1-11 expression (mRNA) data was extracted from the Cancer Genome Atlas (TCGA) HNSCC dataset (n=499). Kaplan-Meier, logrank, and Cox proportional hazards tests were conducted. Levels of α1,2-fucosylated epitopes H-antigen, Lewis Y (LeY), and Lewis B (LeB) were measured in normal, dysplastic, and H&N tumor cell lines via flow cytometry, and RT-qPCR was performed to assess FUT expression. To identify fucosylated signaling and adhesion molecules, fucosylated glycopeptides were enriched from CAL27 and HSC-3 tongue squamous cell carcinoma (SCC) lysates via AAL lectin, followed by nLC-MS/MS. EGFR signaling was assessed via western blot, and parallel plate flow assays were performed to assess in vitro metastatic potential, in tumor cell lines with differential FUT expression. Results: In TCGA analyses, the median survival time (MST) of the FUT2 high expression group was 4.7 years, while low expression group MST was 2.7 years, with an adjusted hazard ratio (aHR) of 0.72 (CI = 0.54-0.95). The FUT6-high group MST was 4.7 years versus 2.9 years for low expression (aHR = 0.62, CI = 0.47-0.83). The FUT7-high group MST was 4.7 years versus 3.5 years for low expression (aHR = 0.72, CI = 0.54-0.95). Expression of the LeY epitope, with both α1,2- and α1,3-linked fucose, varied widely in tongue SCC lines, while pharyngeal SCC lines FaDu and Det-562 displayed intermediate LeY levels, via flow cytometry. EGFR, CD44, and integrins, were among the fucosylated glycoproteins identified via mass spectrometry. HSC-3 cells, sorted into low- and high-LeY populations, demonstrated differential EGFR Tyr-1086 phosphorylation, and differences in adhesion to endothelial cells under flow conditions (in vitro), with implications for tumor metastasis. Conclusion: Overexpression of FUT2, FUT6, and FUT7 were associated with statistically significant increases in survivorship. FUT6 and FUT7 are prominently expressed in immune cells, while FUT2 is expressed in endoderm-derived epithelium. Therefore, FUT6 and FUT7 expression may indicate immune cell infiltration into tumor tissue. Future studies will seek to sort tumor tissue based on cell type, to gain an understanding of the cell-type specific glycosyltransferase expression in HNSCC tumors. Our current results will serve as the foundation to interrogate the role of fucosylated glycoproteins in HNSCC metastasis. Citation Format: Kevin Brown Chandler, Brittany Montesino, Nan Hu, Juan M. Lozano, Robert Sackstein. Fucosyltransferase expression is associated with head and neck cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2400.

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.

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.

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.