Inhibitor Of O-glycosylation Research Articles

Targeting tumor O-glycosylation modulates cancer-immune-cell crosstalk and enhances anti-PD-1 immunotherapy in head and neck cancer.

Cells in the tumor microenvironment (TME) communicate via membrane-bound and secreted proteins, which are mostly glycosylated. Altered glycomes of malignant tumors influence behaviors of stromal cells. In this study, we showed that the loss of core-1 β1,3-galactosyltransferase (C1GALT1)-mediated O-glycosylation suppressed tumor growth in syngeneic head and neck cancer mouse models. O-glycan truncation in tumor cells promoted the M1 polarization of macrophages, enhanced T-cell-mediated cytotoxicity, and reduced interleukin-6 (IL-6) levels in the secretome. Proteasomal degradation of IL-6 was controlled by the O-glycan at threonine 166. Both IL-6/IL-6R blockade and O-glycan truncation in tumor cells induced similar pro-inflammatory phenotypes in macrophages and cytotoxic T lymphocytes (CTLs). The combination of the O-glycosylation inhibitor itraconazole and anti-programmed cell death protein 1 (anti-PD-1) antibody effectively suppressed tumor growth in vivo. Collectively, our findings demonstrate that O-glycosylation in tumor cells governs their crosstalk with macrophages and CTLs. Thus, targeting O-glycosylation successfully reshapes the TME and consequently enhances the efficacy of anti-PD-1 therapy.

Dysfunctional O-glycosylation exacerbates LPS-induced ARDS in mice through impairment of podoplanin expression on alveolar macrophages

O-glycosylation is one of the most common post-translational modifications of proteins, which participates in immune cell recognition, activation, and surveillance. Alveolar macrophage (AM) is closely involved in the pathogenesis of acute respiratory distress syndrome (ARDS), which is associated with excessive, uncontrolled inflammatory response. It is unclear whether and how O-glycosylation affects AM activation as well as ARDS. Here, we administrated experimental mice with an O-glycosylation inhibitor (benzyl-α-GalNAc) at 5 or 10 mg/kg 24 h before intratracheal instillation of LPS (1 mg/kg). At 6 h and 12 h after LPS challenge, mice were sacrificed to collect samples for further analysis. The results showed that benzyl-α − GalNAc induced abundant expression of the Tn antigen, which is a typical marker for abnormal O-glycosylation. Benzyl-α-GalNAc pretreatment significantly exacerbated LPS-induced inflammatory damage in lungs, evidenced by more severe histological signs and drastic elevation of multiple inflammatory cytokines. Mechanistically, benzyl-α-GalNAc impaired podoplanin expression on AMs in mice, which is a heavily O-glycosylated protein that mediates anti-inflammation responses, whereas adoptive transfer of podoplanin-expressing macrophages alleviated LPS-induced ARDS in mice. This study demonstrated that benzyl-α-GalNAc-induced inhibition of O-glycosylation aggravated LPS-induced ARDS in mice, which may be due to down-regulation of podoplanin expression on AMs. We therefore suggest that podoplanin on AMs is essential for lung protection.

A Truncated Mutant – The Most Common Sucrase‐Isomaltase Deficiency in the Inuit Population: Cellular and Biochemical Characterization

Sucrase isomaltase (SI) is the most prominent disaccharidase in the small intestine responsible for the final steps of carbohydrate digestion. Mutations in the SI gene can lead to a drastic reduction or loss of the catalytic activity required for digestion of disaccharides, and thus can be associated with maldigestion and malabsorption of carbohydrates. Carbohydrate malabsorption is one of the most common gastrointestinal problems, where 70% of the population are affected. Congenital sucrase‐isomaltase deficiency (CSID) is an autosomal recessive disorder caused by defective SI activity; and it is clinically characterized by abdominal pain, flatulence, bloating, and diarrhea. Recently, a homozygous frameshift mutation, c.273_274delAG (p.Gly92Leufs*8), was identified in the Inuit population with an observed allele frequency of 17.2%. In this study, Cos‐1 cells were transiently transfected with cDNA encoding the Gly92Leufs*8 mutation or SI wild‐type (SI‐WT) as a control. The assessment of this new variant phenotype was investigated through enzymatic activity measurements, cellular localization and trafficking behavior. The truncation is associated with the elimination of all N‐glycosylation sites, whereas the Ser/Thr‐rich stalk region is retained pointing to a potential O‐glycosylation. In fact, treatment with benzyl 2‐acetamido‐2‐deoxy‐α‐D‐galactopyranoside, an inhibitor of O‐glycosylation in the Golgi, resulted in a substantial shift in the size of the mutant concomitant with transport competence of the mutant to O‐glycosylation in the Golgi Apparatus. The mutant is further trafficked to the cell surface as assessed by the detection of biotinylated forms of the mutant at the cell surface. This result was also corroborated by immunofluorescence images, which revealed the mutant at the cell surface. However, this mutant does not exhibit SI activity due to the absence of the sucrase and isomaltase domains. While our data unraveled the molecular basis for the onset of the clinical symptoms in patients homozygous to this mutation, further work is needed to determine whether heterozygotes with this mutation are also affected and to which extent.

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Inhibition of O-glycosylation aggravates GalN/LPS-induced liver injury through activation of ER stress

Objective O-glycosylation is the most common post-translational modification of proteins, which is involved in many pathophysiological processes including inflammation. Acute liver injury is characterized by an excessive, uncontrolled inflammatory response, but the effects of aberrant O-glycosylation on acute liver injury are yet to explore. Here we aimed to investigate the role of defective O-glycosylation in D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced acute liver damage in mice. Material and methods Experimental mice were administrated with an O-glycosylation inhibitor (benzyl-a-GalNac, 5 mg/kg) at 24 h before administration of GalN/LPS. At 12 h after GalN/LPS administration, mice were sacrificed to collect blood and liver samples for further analysis. Results We found that benzyl-a-GalNac treatment-induced abundant expression of Tn antigen, which is an immature O-glycan representing abnormal O-glycosylation. Benzyl-a-GalNac pretreatment exacerbated considerably GalN/LPS-induced liver damage in mice, evidenced by significantly reduced survival rates, more severe histological alterations, and notable elevation of multiple inflammatory cytokines and chemokines. Mechanistically, benzyl-a-GalNac could trigger endoplasmic reticulum (ER) stress in the liver of mice, demonstrated by the elevated expression of glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), both of which are hallmarks for ER stress. Inhibition of ER stress by 4-phenylbutyric acid (4-PBA) markedly abrogated benzyl-a-GalNac-mediated enhanced hepatotoxicity and systemic inflammation in GalN/LPS-treated mice. Conclusions This study demonstrated that inhibition of O-glycosylation caused by benzyl-a-GalNac aggravated GalN/LPS-induced liver damage and systemic inflammation, which may be due to activation of ER stress.

Abstract 3045: Investigating the influence of glycosylation using cellular models of neuroblastoma

Abstract Background: Neuroblastoma is a common childhood malignancy with approximately 800 new cases diagnosed each year in the United States. Treatment of neuroblastoma directly depends on patient risk categories and includes combinations of surgery, radiation therapy, chemotherapy, stem cell therapy, immunotherapy, and retinoid therapy. Various characteristics of cancer in general and some specific to neuroblastoma cell lines contribute to a difficulty in delivering clinically relevant concentrations of therapeutic agents to neuroblastoma masses. Amplification of the MYCN gene in certain neuroblastoma cell lines correlates with increased cellular surface glycosylation levels as well as a worse prognosis for patients. Overexpression of the MUC1 gene is also associated with increased glycosylation, specifically type-O, in various cancers, but the role of MUC1 in neuroblastoma has not been thoroughly defined. The objective of this study was to evaluate the influence of type-O glycosylation on therapeutic uptake in different neuroblastoma cell lines. Methods: The neuroblastoma cell line SK-N-DZ, a MYCN-amplified line, was compared to SK-N-SH, a non-MYCN-amplified line (negative control) and BT-20, a highly glycosylated breast cancer cell line (positive control). Benzyl-α-GalNAc, an O-glycosylation inhibitor, was introduced to each cell line. To determine glycosylation levels in the respective cell lines, fluorescence assays were performed using CD227 (anti-MUC1) antibody. In vitro cytotoxicity assays were performed to determine maximum non-toxic concentrations of the glycosylation inhibitor (benzyl-α-GalNAc) and antibody (CD227) and to subsequently evaluate the functional role of glycosylation using cellular models of neuroblastoma. Results: The maximum non-toxic concentrations per 10,000 cells for each neuroblastoma cell line was 0.05 mg/ml of benzyl-α-GalNAc, and 6 µl of CD227 stock preparation. Preliminary results from fluorescence detection studies showed an increase in CD227-FITC antibody uptake by the MYCN-amplified neuroblastoma cells (SK-N-DZ) following exposure to the O-glycosylation inhibitor (benzyl-α-GalNAc). Cellular uptake of CD227-FITC by non-MYCN-amplified cell line (SK-N-SH) was less by comparison. Conclusion: Preliminary findings suggest that O-glycosylation is a barrier capable of limiting cellular uptake of agents in MYCN-amplified neuroblastoma cell lines. Ongoing studies are focused on investigating the influence of glycosylation inhibition on cytotoxicity of FDA-approved therapeutic agents. Citation Format: Meghan Cook, Alma Garcia, Katie Konieczny, Meghan Ferguson, Robert Campbell. Investigating the influence of glycosylation using cellular models of neuroblastoma [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 3045.

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac5GalNTGc

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac5GalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blotanalysis of HL-60 promyelocytes demonstrated that 50-80μM Ac5GalNTGc prevented elaboration of 30%-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.

Deficiency of O-linked-glycosylation regulates activation of T cells and aggravates Concanavalin A-induced liver injury

ObjectiveProtein glycosylation is involved in immunological recognition and immune cell activation. The role of O-glycosylation in Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) was elucidated in the present study. MethodsMice were intravenously injected with Con A (10 mg/kg) to establish an AIH mouse model. Here, 24 h prior to administration of Con A, experimental mice were intragastrically administrated with O-glycosylation inhibitor (benzyl-α-GalNAc) at doses of 1 and 5 mg/kg, respectively, while control mice were administrated with the same volume of saline. Before and after administration of Con A for 6 and 12 h, mice were sacrificed and their plasma and livers were collected to score liver injury. Peripheral blood, spleen, and thymus were collected for flow cytometry analysis. The expression levels of neutrophilic alkaline phosphatase-3 (NALP3) and NALP6 in liver were evaluated as well. ResultsPre-treatment with benzyl-α-GalNAc increased the serum transaminase levels and induced more infiltration and necrosis in livers of Con A administrated mice. The levels of some pro-inflammation cytokines also increased in administrated mice. In addition, pretreatment with benzyl-α-GalNAc up-regulated the expression levels of NALP3 and NALP6. And benzyl-α-GalNAc inhibited the levels of apoptosis of thymus cells and influenced activation of T cells in peripheral blood and spleen of Con A administrated mice, especially that accelerated the physiological progression of CD4+CD25−CD69+ subset. ConclusionThe present research demonstrated that benzyl-α-GalNAc aggravated Con A-induced AIH, and the role of the O-glycosylation inhibitor as the aggravation may be related to regulation of the levels of cytokines, as well as influencing proliferation of T cells.

O-GlcNAc Signaling Augmentation Protects Human Corneal Endothelial Cells from Oxidative Stress via AKT Pathway Activation

ABSTRACTPurpose: To investigate the effect of inhibitor of O-glycosylation on human corneal endothelial cells (HCECs) under oxidative stress.Methods: HCECs were cultured and treated with 10 mM tert-butyl hydroperoxide (tBHP) with or without PUGNAc, a known inhibitor of OGA. Cell viability was assessed. Mitochondrial membrane potential (ΔΨm) was measured. Intracellular Ca2+ levels and mitochondrial Ca2+ levels were measured. Intracellular reactive oxygen species formation was measured. Levels of O-linked β-N-acetylglucosamine (O-GlcNAc), AKT, and pAKT were evaluated by Western blotting.Results: O-GlcNAc augmentation by PUGNAc increased cell viability, attenuated the loss of ΔΨm, and intracellular ROS against tBHP-induced oxidative stress (p < .05). O-GlcNAc augmentation reduced tBHP-induced mitochondrial calcium overload (p < .05) while it did not have any effect on intracellular calcium overload with tBHP. Furthermore, AKT signaling was activated in the cells with O-GlcNAc augmentation.Conclusions: O-GlcNAc signaling augmentation protects HCECs from oxidative stress via activation of AKT pathways.

Ridge regression estimated linear probability model predictions of O-glycosylation in proteins with structural and sequence data

BackgroundTo-date, no claim regarding finding a consensus sequon for O-glycosylation has been made. Thus, predicting the likelihood of O-glycosylation with sequence and structural information using classical regression analysis is quite difficult. In particular, if a binary response is used to distinguish between O-glycosylated and non-O-glycosylated sequences, an appropriate set of non-O-glycosylatable sequences is hard to find.ResultsThree sequences from similar post-translational modifications (PTMs) of proteins occurring at, or very near, the S/T-site are analyzed: N-glycosylation, O-mucin type (O-GalNAc) glycosylation, and phosphorylation. Results found include: 1) The consensus composite sequon for O-glycosylation is: ~(W–S/T–W), where “~” denotes the “not” operator. 2) The consensus sequon for phosphorylation is ~(W–S/T/Y/H–W); although W–S/T/Y/H–W is not an absolute inhibitor of phosphorylation. 3) For linear probability model (LPM) estimation, N-glycosylated sequences are good approximations to non-O-glycosylatable sequences; although N – ~P – S/T is not an absolute inhibitor of O-glycosylation. 4) The selective positioning of an amino acid along the sequence, differentiates the PTMs of proteins. 5) Some N-glycosylated sequences are also phosphorylated at the S/T-site in the N – ~P – S/T sequon. 6) ASA values for N-glycosylated sequences are stochastically larger than those for O-GlcNAc glycosylated sequences. 7) Structural attributes (beta turn II, II´, helix, beta bridges, beta hairpin, and the phi angle) are significant LPM predictors of O-GlcNAc glycosylation. The LPM with sequence and structural data as explanatory variables yields a Kolmogorov-Smirnov (KS) statistic of 99%. 8) With only sequence data, the KS statistic erodes to 80%, and 21% of out-of-sample O-GlcNAc glycosylated sequences are mispredicted as not being glycosylated. The 95% confidence interval around this mispredictions rate is 16% to 26%.ConclusionsThe data indicates the existence of a consensus sequon for O-glycosylation; and underscores the germaneness of structural information for predicting the likelihood of O-glycosylation.

Morniga-G, a T/Tn-Specific Lectin, Induces Leukemic Cell Death via Caspase and DR5 Receptor-Dependent Pathways.

Morniga-G, the Gal-specific black mulberry (Morus nigra) lectin, displays high affinity for T (CD176) and Tn (CD175) antigens, frequently expressed at the cancer cell surface. The effects of Morniga-G were investigated on a Tn-positive leukemic Jurkat cell line. The lectin, used in a concentration range between 5–20 μg/mL, induced cell death in leukemic Jurkat cells. Microscopic and cytofluorometric analyses indicated that Jurkat cell death was essentially apoptotic, associated with an increase in the ceramide content and a depolarization of the mitochondrial transmembrane potential. This lectin-mediated cell death was inhibited by the pan caspase-inhibitor zVAD. In addition, cleavage of caspases 8, 9, and 3 was observed in Morniga-G-treated Jurkat cells whereas Jurkat cell lines that are deficient in caspase 8–10, caspase 9, or FADD, survived to the lectin-mediated toxicity. Furthermore, in the presence of TRAIL- or DR5-blocking mononoclonal antibodies, Jurkat cells became resistant to Morniga-G, suggesting that the lectin triggers cell death via the TRAIL/DR5 pathway. In silico computer simulations suggest that Morniga-G might facilitate both the DR5 dimerization and the building of TRAIL/DR5 complexes. Finally, upon treatment of Jurkat cells with benzyl-GalNAc, an O-glycosylation inhibitor, a decrease in Tn antigen expression associating with a reduced Morniga-G toxicity, was observed. Taken together, these results suggest that Morniga-G induces the cell death of Tn-positive leukemic cells via concomitant O-glycosylation-, caspase-, and TRAIL/DR5-dependent pathways.

Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: Dependency on integrin and the Rho GTPase family.

To determine the biological roles of cell surface glycosylation, we modified the surface glycosylation of human malignant lymphoma cell lines using glycosylation inhibitors. The O-glycosylation inhibitor, benzyl-α-GalNAc (BZ) enhanced the fibronectin adhesion of HBL-8 cells, a human Burkitt's lymphoma cell line, and of H-ALCL cells, a human anaplastic large cell lymphoma cell line, both of which were established in our laboratory. The N-glycosylation inhibitor, tunicamycin (TM) inhibited the surface expression of Phaseolus vulgaris leukoagglutinating (L-PHA) lectin- and Canavalia ensiformis (ConA) lectin-reactive oligosaccharides in the HBL-8 cell line. Assay of the adhesion of HBL-8 cells to fibronectin showed that fibronectin adhesion is mediated by the integrin very late antigen (VLA)-4 and that not only BZ but also TM treatment enhanced HBL-8 cell adhesion to fibronectin. Furthermore, although BZ treatment also enhanced H-ALCL cell adhesion to fibronectin, this effect was not mediated by VLA-5 or the RGD sequence of fibronectin. We also showed that H-ALCL cell adhesion to galectin-3 was enhanced by pre-treatment with neuraminidase, which cleaves cell surface sialic acid. Additionally, H-ALCL cell adhesion to galectin-3 was inhibited by pre-treatment with the RGD peptide suggesting that cell adhesion to galectin-3 is mediated by integrin (VLA-5). Furthermore, H-ALCL cell invasion of galectin-1 and galectin-3 was inhibited by pre-treatment with the RGD peptide. Therefore, cell adhesion to and invasion of galectin-1 and galectin-3 are integrin-dependent. In addition to these findings, cell adhesion to galectin-3 was markedly inhibited by treatment with β-lactose compared to treatment with sucrose. Therefore, interactions between integrins and galectin-3 may be mediated through β-galactose that is linked to glycans of integrins. AZA1, an inhibitor of Ras homolog oncoprotein (Rho) GTPase family proteins, RAS-related C3 botulinus toxin substrate 1 (Rac 1) and Cell division control protein 42 homolog (Cdc42) markedly inhibited cell invasion of galectin-1 and galectin-3 suggesting that Rac 1 and Cdc42 may be involved in the regulation of H-ALCL cell invasion of galectins. In conclusion, artificial modification of cell surface glycosylation revealed the biological roles of glycosylation in the adhesion to and invasion of the extracellular matrix (ECM) by human malignant lymphoma cell lines. These findings will provide new insight into the glycobiology of human malignant lymphoma.

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion

Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.

Abstract 3226: GALNT6 stabilizes GRP78 protein by O-type glycosylation

Abstract Aberrant O-type glycosylation is predominantly detected in breast cancer and plays diverse roles in cancer cell proliferation, metastasis, and drug resistance. GALNT (polypeptide N-acetylgalactosaminyl transferase) family initiates the O-type glycosylation and its high activity accounts for aberrant O-type glycosylation in breast cancer. We previously suggested that GALNT6 is a promising molecular target in breast cancer and involved in O-type glycosylation of Mucin 1 and fibronectin. To further investigate the molecular function of GALNT6, we performed pull down assay using VVA (Vicia Villosa agglutinin) lectin and identified several O-glycosylated proteins specifically in GALNT6-positive stable cells. After in vitro and in vivo O-glycosylation assays, we confirmed GRP78 (glucose related protein 78) as a physiological substrate of GALNT6 and identified 5 O-glycosylation sites in GRP78 by mass spectrometry. GRP78 is a molecular chaperone that facilitates proper protein folding under cellular stress. Interestingly, accumulating evidences have indicated that GRP78 plays anti-apoptotic roles in cancer cells against chemotherapy. Exogenous induction of GALNT6 increased the amount of GRP78 protein without any changes in its transcriptional level. Concordantly, knockdown of GALNT6 significantly reduced the GRP78 protein. Collectively, our findings imply that GALNT6 stabilizes GRP78 via O-glycosylation and GALNT6 inhibitor will be able to sensitize breast cancer cells to chemotherapy. Citation Format: Jae-Hyun Park, Suyoun Chung, Jiaying Lin, Koji Ueda, Toyomasa Katagiri, Yusuke Nakamura. GALNT6 stabilizes GRP78 protein by O-type glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3226. doi:10.1158/1538-7445.AM2014-3226

Abstract 4469: An evaluation of the role of mucin in nano drug delivery

Abstract Introduction: Solid tumors comprise more than 85% of cancers that affect humans. The presence of the extracellular matrix, namely mucin, on the tumor cell is considered one of the barriers against effective treatment of solid tumors. In tumor cells, mucin is aberrantly overexpressed producing a mucin mesh which may impede the rate of entry of chemotherapy medications. The type O-glycosylation inhibitor, benzyl-α-GalNAc, significantly reduces mucin type O-glycosylation in mucin-producing tumor cells resulting in enhanced cytotoxic drug action. We next seek to determine the role of mucin in the delivery of nano-sized drug delivery systems. Methods: The cell lines used were derived from tumor or normal tissues representing multiple organ environments. These cell lines include: Capan-1, Capan-2, PANC-1, HPAF-II (human adenocarcinoma), hTERT-HPNE (normal human pancreas), CRL-1777 (normal murine pancreas), ZR-75-1 (breast cancer), and CRL-2089 (normal breast). Evaluation of MUC1 protein expression was done by western blot analysis in addition to fluorescence microplate reader using CD227 (anti-MUC1 antibody). Furthermore, we investigated the cellular uptake of the fluorescently labeled polystyrene microsphere beads and liposomes in the different cell lines. Finally, FACS analysis was used to determine cellular uptake following inhibition of mucin type O-glycosylation and compared to control. Results: Different levels of mucin expression were observed among the various cell lines. By investigating the antibody association with the MUC1 protein core, Capan-1and ZR-75-1have shown the greatest cellular uptake following inhibition of glycosylation. To evaluate the cellular uptake of the microsphere beads and liposomes we used cell lines derived from the pancreas. The glycosylation inhibitor had little to no effect on the uptake of the microsphere beads, regardless of cell line employed. However, the overall uptake of the large beads (990 nm) was significantly greater in comparison to the uptake of the smaller beads (67 and 116 nm). Next, we carried out the same procedure using liposomes that were made in the size range of 60-80nm, 100-120nm, and 350-550nm. Out of the 6 pancreatic cell lines examined, PANC-1 cell line showed that the uptake of each liposome preparation type was significantly greater following the inhibition of mucin glycosylation. In addition, we observed that the uptake of liposomes increased as a function of liposome size. This was confirmed by FACS analysis as well. Conclusion: Our results suggest that inhibiting the mucin type O- glycosylation alters the uptake of the microsphere beads in all the cell lines examined. As for the liposomes, however, the effect of inhibiting glycosylation is cell line dependent. Further studies will be performed to investigate the factors underlying the differential uptake observed with liposomes following inhibition of glycosylation. This might be helpful in developing a better nano-therapeutic approach to treat cancer. Citation Format: Musaed A. Alkholief, Robert Campbell. An evaluation of the role of mucin in nano drug delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4469. doi:10.1158/1538-7445.AM2014-4469

Pivotal role of MUC1 glycosylation by cigarette smoke in modulating disruption of airway adherens junctions in vitro.

Cigarette smoke increases the risk of lung cancer by 20-fold and accounts for 87% of lung cancer deaths. In the normal airway, heavily O-glycosylated mucin-1 (MUC1) and adherens junctions (AJs) establish a structural barrier that protects the airway from infectious, inflammatory and noxious stimuli. Smoke disrupts cell-cell adhesion via its damaging effects on the AJ protein epithelial cadherin (E-cad). Loss of E-cad is a major hallmark of epithelial-mesenchymal transition (EMT) and has been reported in lung cancer, where it is associated with invasion, metastasis and poor prognosis. Using organotypic cultures of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk), we have demonstrated that E-cad loss is regulated through the aberrant interaction of its AJ binding partner, p120-catenin (p120ctn), and the C-terminus of MUC1 (MUC1-C). Here, we reported that even before MUC1-C became bound to p120ctn, smoke promoted the generation of a novel 400 kDa glycoform of MUC1's N-terminus (MUC1-N) differing from the 230 kDa and 150 kDa glycoforms in untreated control cells. The subsequent smoke-induced, time-dependent shedding of glycosylated MUC1-N exposed MUC1-C as a putative receptor for interactions with EGFR, Src and p120ctn. Smoke-induced MUC1-C glycosylation modulated MUC1-C tyrosine phosphorylation (TyrP) that was essential for MUC1-C/p120ctn interaction through dose-dependent bridging of Src/MUC1-C/galectin-3/EGFR signalosomes. Chemical deglycosylation of MUC1 using a mixture of N-glycosylation inhibitor tunicamycin and O-glycosylation inhibitor benzyl-α-GalNAc disrupted the Src/MUC1-C/galectin-3/EGFR complexes and thereby abolished smoke-induced MUC1-C-TyrP and MUC1-C/p120ctn interaction. Similarly, inhibition of smoke-induced MUC1-N glycosylation using adenoviral shRNA directed against N-acetyl-galactosaminyl transferase-6 (GALNT6, an enzyme that controls the initiating step of O-glycosylation) successfully suppressed MUC1-C/p120ctn interaction, prevented E-cad degradation and maintained cellular polarity in response to smoke. Thus, GALNT6 shRNA represents a potential therapeutic modality to prevent the initiation of events associated with EMT in the smoker's airway.

Inhibition of KL-6/MUC1 glycosylation limits aggressive progression of pancreatic cancer.

To evaluate the significance of KL-6/MUC1 (a type of MUC1) glycosylation in pancreatic cancer progression. KL-6/MUC1 expression was detected by immunohistochemistry in 48 patients with pancreatic duct cell carcinoma. The N-/O-glycosylation inhibitors (tunicamycin and benzyl-N-acetyl-α-galactosaminide) were then used to interfere with KL-6/MUC1 glycosylation in two pancreatic carcinoma cell lines, and the effects on KL-6/MUC1 expression, and cell adhesion and invasion were determined. In addition, protein expression of epithelial-mesenchymal transition markers, E-cadherin and vimentin, were evaluated in cells after treatment with glycosylation inhibitors. Overexpression of KL-6/MUC1 was found in all pancreatic cancer tissues, but not in the surrounding normal pancreatic tissues. The expression profile of KL-6/MUC1 was significantly decreased after treatment with the inhibitors. The adhesion and invasive ability of cancer cells were significantly decreased after drug treatment, and increased E-cadherin and decreased vimentin expression were found. KL-6/MUC1 glycosylation is involved in pancreatic cancer metastasis and invasion. Therapeutic strategies which target this may help control the aggressive behavior of pancreatic cancer cells.

Abstract 4511: An evaluation of the role of Mucin in nano drug delivery.

Abstract Introduction: Solid tumors comprise more than 85% of cancers that affect humans. The presence of the extracellular matrix, namely mucin, on the tumor cell is considered one of the barriers against effective treatment of solid tumors. In tumor cells, mucin is aberrantly overexpressed producing a mucin mesh which may impede the rate of entry of chemotherapy medications. The type O-glycosylation inhibitor, benzyl-α-GalNAc, significantly reduces mucin type O-glycosylation in mucin-producing tumor cells resulting in enhanced cytotoxic drug action. We next seek to determine the role of mucin in the delivery of nano-sized drug delivery systems. Methods: The cell lines used in our studies were derived from tumor or normal tissues representing multiple different tissue environments. A fluorescence microplate reader was used to evaluate the MUC1 expression in the cell lines by using CD227 (anti-MUC1 antibody). We investigated the cellular uptake of the fluorescently labeled polystyrene microsphere beads in Capan-1 (a human pancreatic cancer cell line). The size of these beads was determined by particle size analysis. FACS analysis was used to determine cellular uptake following inhibition of mucin type O-glycosylation and compared to control. Finally, fluorescence microscopy was used to show the effect of inhibitor on the uptake of the beads into ZR-75-1 (a human breast cancer cell line) and Capan-1 cells. Results: Different levels of mucin expression were observed among the various cell lines. By investigating the antibody association with the MUC1, Capan-1and ZR-75-1have shown the greatest cellular uptake following inhibition of glycosylation. The microspheres used in this study were 920 ±70 nm, 100 ±36 nm, and 52 ±8 nm in size. Cellular uptake studies of the microsphere beads into Capan-1 showed a significant increase in the uptake of the 920 nm beads in cells when compared to control cells. However, in case of the smaller beads, we did not observe a significant increase of the cellular uptake following inhibition of the mucin glycosylation. FACS analysis of the beads with Capan-1 cell line showed a red shift in the fluorescence peak of the 920 nm beads in cells treated with inhibitor compared to control. The smaller beads (52 nm and 100 nm), however, did not show any shift indicating that the uptake of the beads was hindered by the mucin mesh. Conclusion: Our results suggest that inhibiting the mucin type O-glycosylation alters the uptake of relatively large microsphere beads. This might be helpful in developing a better therapeutic approach to treat cancer. Citation Format: Musaed Allkholief, Robert Campbell. An evaluation of the role of Mucin in nano drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2013-4511

FcμR (TOSO/FAIM3) Is An O-Glycosylated Endocytic Receptor That Shuttles IgM From the Cell Surface to the Lysosome and Whose Expression Is Regulated by TLR Signaling

Abstract 2828TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcμR). Previous studies have shown that FcμR is selectively overexpressed in chronic lymphocytic leukemia (CLL) cells as compared to normal B-cells or other B cell malignancies. In this study, we extend the characterization of FcμR and identify the mechanisms regulating its trafficking and expression. In CLL cells, analysis of FcμR protein expression by immunoblotting revealed one major band around 60 kDa, a series of fainter migrating species, and a band at 41 kDa corresponding to its predicted molecular weight. By contrast, normal B cells express predominantly the 60 kDa form. Inspection of FcμR sequence revealed numerous potential O-glycosylation sites in the extracellular domain and treatment with a general inhibitor of O-glycosylation, benzyl-N-acetyl- α-galactosaminide, confirmed that FcμR is indeed heavily O-glycosylated. By immunofluorescence confocal microscopy, we determined that O-glycosylation is critical for trafficking of FcμR to the cell surface as mutations in the predicted O-glycosylation sites led to intracellular retention of FcμR in HeLa cells transfected with the appropriate expression vectors. In addition, pharmacologic inhibition of O-glycosylation decreased FcμR expression on CLL cells. Next, we used biotinylated IgM and streptavidin DyLight 488 to determine the fate of IgM bound to FcμR. Addition of IgM to CLL cells prompted rapid internalization of both IgM and FcμR, reaching half maximal internalization of cell bound IgM within one minute and virtually complete internalization within 5 minutes. By contrast, a monoclonal antibody specific for FcμR was not internalized. Using immunofluorescence confocal microscopy and co-staining with transferrin and anti-LAMP-1 antiserum, we followed IgM trafficking. FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. This last step was chloroquine but not bortezomib sensitive, confirming the role of the lysosome in FcμR degradation. Using a series of FcμR deletion mutants, we identified a proline-rich domain that is essential for cell surface expression of FcμR and a second domain, containing a YXX Φ motif, that controls internalization.Of particular interest, but so far elusive, is a possible functional role of FcμR in CLL pathogenesis. Our data suggest that a major physiologic role of FcμR may be internalization of IgM bound cargo into cells. Given the broad reactivity of IgM such cargo likely includes a variety of infectious agents as well as cellular debris that will be transported into the lysosome and thereby brought into contact with intracellular Toll-like Receptors (TLRs). To address a possible interaction between FcμR and TLRs, we investigated the effect of TLR activation on FcμR expression. There was a striking downregulation of FcμR on CLL cells in response to activation of TLR7 (using imiquimod) or TLR9 (using CpG-ODN) that involved both inhibition of transcription as well as degradation of FcμR through the lysosome. Following CpG stimulation, FcμR mRNA decreased with a half life of approximately 3 hours; that is at the same rate as after actinomycin D treatment. At 24 hours, FcμR protein was greatly reduced in response to either CpG or imiquimod, but interestingly there was less response to CpG in IGHV unmutated than in IGHV mutated CLL samples (p<0.001). In summary, FcμR is an O-glycosylated endocytic receptor that shuttles IgM from the cell surface to the lysosome. In vivo, FcμR could play a role in presenting IgM opsonized immune complexes to intracellular TLRs resulting in B-cell activation. Further studies are needed to clarify a potential contribution of FcμR to the activation of CLL cells in vivo. In addition, owing to the rapid internalization of IgM, we are pursuing FcμR as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells (to be presented in a separate abstract).This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute. Disclosures:No relevant conflicts of interest to declare.

Effect of O-Glycosylation and Sialylation Inhibitors on Classical NLS-Dependent Nuclear Protein Import in HT29-MTX Human Colon Cancer Cells

Glycosylation, including O-linked, mucin type, has an import role in the function and activity of many proteins, particularly those that are secreted or transcellular. Previous studies have indicated that mucin-type O-linked glycans may also be carried by intracellular proteins such as the stress-related protein Orp150 that is involved in classical nuclear localization sequence (NLS)-dependent nuclear protein import. This study investigated the influence on NLS-dependent nuclear protein import of the potent O-glycosylation inhibitor, Benzyl-GalNAc; the sialylation inhibitor, 5’CDP; the Golgi proton pump inhibitor, bafilomycin; and the pro-inflammatory cytokine, TNFα. Treatment of the mucus-secreting human colon cancer HT29-MTX cells with each of these agents caused a global increase of the cellular core 1 carbohydrate structure (galactose β1,3 N-acetylgalactosamine), one of the precursor structures of the complex mucin-type glycans. Benzyl-GalNAc treatment also increased the expression of the core 1 structure on cell surface glycoconjugates and caused a generalised decrease in sialylation. Hsp70 nuclear translocation upon heat stress, a process that is mediated by the classical NLS pathway, was, however, not affected by pre-treatment of the cells with any of these agents. This suggests that O-linked and/or sialylated glycans are unlikely to be involved in the classical NLS-dependent protein import mechanism.