RESUMEN
Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.1E12 is a promising anti-MUC1 antibody with a distinct specificity toward MUC1 modified with an immature O-glycan (NeuAcα(2-3)Galß(1-3)GalNAc) on a specific Thr. However, the structural basis for the interaction between MY.1E12 and MUC1 remains unclear. The aim of this study is to elucidate the mode of interaction between MY.1E12 and MUC1 O-glycopeptide by NMR, molecular dynamics (MD) and docking simulations. NMR titration using MUC1 O-glycopeptides suggests that the epitope is located within the O-linked glycan and near the O-glycosylation site. MD simulations of MUC1 glycopeptide showed that the O-glycosylation significantly limits the flexibility of the peptide backbone and side chain of the O-glycosylated Thr. Docking simulations using modeled MY.1E12 Fv and MUC1 O-glycopeptide, suggest that VH mainly contributes to the recognition of the MUC1 peptide portion while VL mainly binds to the O-glycan part. The VH/VL-shared recognition mode of this antibody may be used as a template for the rational design and development of anti-glycopeptide antibodies.
Asunto(s)
Glicopéptidos , Simulación de Dinámica Molecular , Anticuerpos Monoclonales , Glicopéptidos/química , Espectroscopía de Resonancia Magnética , Mucina-1/metabolismo , Polisacáridos/químicaRESUMEN
Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.
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Antineoplásicos Inmunológicos , Carcinoma de Células Escamosas , Animales , Anticuerpos Monoclonales , Células CHO , Cricetinae , Cricetulus , Ratones , Mucina-1/química , Mucinas/química , Polisacáridos/químicaRESUMEN
Ligand-induced cellular signaling involved in interleukin 10 (IL-10) production by lamina propria macrophages (LPMs) during their interactions with commensal bacteria is not clearly understood. We previously showed, using mice lacking a C-type lectin MGL1/CD301a, that this molecule on colonic LPMs plays an important role in the induction of IL-10 upon interaction with commensal bacteria, Streptococcus sp. In the present report, we show that the physical engagement of MGL1/CD301a on LPMs with in-situ isolated Streptococcus sp. bacteria leads to IL-10 messenger RNA (mRNA) induction. Spleen tyrosine kinase (Syk), caspase recruitment domain 9 (CARD9) and extracellular signal-regulated kinase (ERK), but not NF-κB pathway, are shown to be indispensable for IL-10 mRNA induction after stimulation with heat-killed Streptococcus sp. Guanidine hydrochloride treatment of Streptococcus sp., which is known to extract bacterial cell surface glycan-rich components, abolished bacterial binding to recombinant MGL1/CD301a. The extract contained materials which bound rMGL1 in ELISA and appeared to induce IL-10 mRNA expression in LPMs in vitro. Lectin blotting showed that the extract contained glycoproteins that are considered as putative ligands for MGL1. Some human commensal Lactobacillus species also induced IL-10 mRNA expression by colonic LPMs in vitro, which depends on the presence of MGL1/CD301a and CARD9. The present results are the first to show that MGL1/CD301a acts as a signal transducer during colonic host-microbe interactions.
Asunto(s)
Asialoglicoproteínas , Interleucina-10 , Animales , Asialoglicoproteínas/genética , Asialoglicoproteínas/metabolismo , Bacterias/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Interleucina-10/genética , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Decreased cell adhesion has been reported as a significant negative prognostic factor of lung cancer. However, the molecular mechanisms responsible for the cell incohesiveness in lung cancer have not yet been elucidated in detail. We herein describe a rare histological variant of lung adenocarcinoma consisting almost entirely of individual cancer cells spreading in alveolar spaces in an incohesive pattern. A whole exome analysis of this case showed no genomic abnormalities in CDH1 or other genes encoding cell adhesion molecules. However, whole mRNA sequencing revealed that this case had an extremely high expression level of mucin 21 (MUC21), a mucin molecule that was previously shown to inhibit cell-cell and cell-matrix adhesion. The strong membranous expression of MUC21 was found on cancer cells using mAbs recognizing different O-glycosylated forms of MUC21. An immunohistochemical analysis of an unselected series of lung adenocarcinoma confirmed that the strong membranous expression of MUC21 correlated with incohesiveness. Thus, MUC21 could be a promising biomarker with potential diagnostic and therapeutic applications for lung adenocarcinoma showing cell incohesiveness.
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Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Adenocarcinoma del Pulmón/diagnóstico por imagen , Adenocarcinoma del Pulmón/genética , Anciano , Antígenos CD/genética , Cadherinas/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/genética , Tomografía Computarizada por Rayos X , Secuenciación del ExomaRESUMEN
Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca(2+)-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca(2+)-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.
Asunto(s)
Asialoglicoproteínas/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Internalización del Virus , Animales , Asialoglicoproteínas/genética , Células CHO , Calcio/metabolismo , Línea Celular , Cricetinae , Cricetulus , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Lectinas Tipo C/genética , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Receptores Virales/genéticaRESUMEN
Glycosylation changes of cancer cells are known to be associated with malignant progression and metastases and potentially determine the organ-selective nature of metastasis as theorized by Paget (Lancet 1:571-573, 1889). Cellular glycans play a variety of roles in the processes of metastasis and may be unique to the cells that metastasize to different organs. We analyzed the glycosylation profiles of the primary tumor and tumors metastasized to lymph node, liver, lung, brain, bone, thyroid, kidney, adrenal, small intestine and pancreas in an autopsy case of breast cancer employing a lectin microarray with 45 lectins. Clustering analysis of the data revealed that metastatic breast cancer cells were categorized into several clusters according to their glycosylation profiles. Our results provide a biological basis to understand differential phenotypes of metastatic breast cancer cells potentially reflecting clonal origin, which does not directly reflect genomic or genetic changes or microenvironmental effects but connects to glycosylation profiles.
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Neoplasias de la Mama , Humanos , Glicosilación , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Metástasis de la Neoplasia , Lectinas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/genéticaRESUMEN
The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.
RESUMEN
Adjuvant chemotherapy has played a major role in the treatment of hormone receptor-positive breast cancer for many years. To better determine which patient subsets need adjuvant chemotherapy, various gene expression analyses have been developed, but cost-effective tools to identify such patients remain elusive. In the present report, we retrospectively investigated immunohistochemical expression and subcellular localization of MUC1 in primary tumors and examined their relationship to tumor malignancy, chemotherapy effect and patient outcomes. We retrospectively examined three patient cohorts with hormone receptor-positive/human epidermal growth factor receptor 2-negative invasive breast cancer: 51 patients who underwent 21-gene expression analysis (multi-gene assay-cohort), 96 patients who received neoadjuvant chemotherapy (neoadjuvant chemotherapy-cohort), and 609 patients whose tumor tissue was used in tissue-microarrays (tissue-microarray-cohort). The immunohistochemical staining pattern of the anti-MUC1 monoclonal antibody, Ma695, was examined in cancer tissues, and subcellular localization was determined as apical, cytoplasmic or negative. In the multi-gene assay-cohort, tumors with apical patterns had the lowest recurrence scores, reflecting lower tumor malignancy, and were significantly lower than MUC1-negative tumors (P = 0.038). In the neoadjuvant chemotherapy-cohort, there was no correlation between MUC1 staining patterns and effects of chemotherapy. Finally, in the tissue-microarray-cohort, we found that patients with apical MUC1 staining patterns had significantly longer disease-free-survival and overall survival than other patterns (P = 0.020 and 0.039, respectively). Our data suggest that an apical MUC1 staining pattern indicates luminal A-likeness. Assessment of the subcellular localization of MUC1 glycoprotein may be useful for identifying patients who can avoid adjuvant chemotherapy.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Estudios Retrospectivos , Mucina-1/metabolismo , Supervivencia sin Enfermedad , Glicoproteínas/uso terapéuticoRESUMEN
BACKGROUND: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. METHODS: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. RESULTS: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. CONCLUSIONS: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Antígeno B7-H1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inmunidad Celular , Células Asesinas Naturales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismoRESUMEN
Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma de Células Escamosas/diagnóstico , Epítopos/análisis , Neoplasias Esofágicas/diagnóstico , Glicoproteínas de Membrana/química , Mucinas/química , Acetilgalactosamina/química , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Células CHO , Secuencia de Carbohidratos , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Cricetinae , Células Epiteliales/química , Células Epiteliales/citología , Epítopos/inmunología , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Esófago/química , Esófago/citología , Citometría de Flujo , Expresión Génica , Glicosilación , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Mucinas/genética , Mucinas/inmunología , TransfecciónRESUMEN
Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.
RESUMEN
Glycosylation is one of the most important post-translational modifications of cell surface proteins involved in the proliferation, metastasis and treatment resistance of cancer cells. However, little is known about the role of glycosylation as the mechanism of breast cancer cell resistance to endocrine therapy. Herein, we aimed to identify the glycan profiles of tamoxifen-resistant human breast cancer cells, and their potential as predictive biomarkers for endocrine therapy. We established tamoxifen-resistant cells from estrogen receptor-positive human breast cancer cell lines, and their membrane-associated proteins were subjected to lectin microarray analysis. To confirm differential lectin binding to cellular glycoproteins, we performed lectin blotting analyses after electrophoretic separation of the glycoproteins. Mass spectrometry of the tryptic peptides of the lectin-bound glycoproteins was further conducted to identify glycoproteins binding to the above lectins. Finally, expression of the glycans that were recognized by a lectin was investigated using clinical samples from patients who received tamoxifen treatment after curative surgery. Lectin microarray analysis revealed that the membrane fractions of tamoxifen-resistant breast cancer cells showed increased binding to Wisteria floribunda agglutinin (WFA) compared to tamoxifen-sensitive cells. Glycoproteins seemed to be responsible for the differential WFA binding and the results of mass spectrometry revealed several membrane glycoproteins, such as CD166 and integrin beta-1, as candidates contributing to increased WFA binding. In clinical samples, strong WFA staining was more frequently observed in patients who had developed distant metastasis during tamoxifen treatment compared with non-relapsed patients. Therefore, glycans recognized by WFA are potentially useful as predictive markers to identify the tamoxifen-resistant and relapse-prone subset of estrogen receptor-positive breast cancer patients.
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Neoplasias de la Mama , Tamoxifeno , Antígenos de Neoplasias , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Glicoproteínas/metabolismo , Humanos , Recurrencia Local de Neoplasia , Lectinas de Plantas/metabolismo , Polisacáridos/metabolismo , Receptores de Estrógenos , Receptores N-Acetilglucosamina/metabolismo , Tamoxifeno/farmacologíaRESUMEN
The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits alpha5, alpha6, and beta1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.
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Regulación de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Aminoácidos/química , Animales , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Glicoproteínas/química , Glicosilación , Humanos , Ratones , Modelos Biológicos , Polisacáridos/química , Estructura Terciaria de ProteínaRESUMEN
Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with alpha-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1(-/-) and Mgl2(-/-) BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than beta-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4(+) T cell activation.
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Células Dendríticas/citología , Lectinas Tipo C/metabolismo , Animales , Antígenos/química , Células de la Médula Ósea/citología , Células CHO , Cricetinae , Cricetulus , Femenino , Citometría de Flujo/métodos , Glicosilación , Inmunohistoquímica/métodos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Linfocitos T/metabolismo , Distribución TisularRESUMEN
Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.
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Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/virología , Lectinas Tipo C/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Sustitución de Aminoácidos , Línea Celular Tumoral , Ebolavirus/genética , Ebolavirus/fisiología , Células HEK293 , Fiebre Hemorrágica Ebola/metabolismo , Humanos , Mutagénesis , Proteínas del Envoltorio Viral/genéticaRESUMEN
Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.
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Acetilglucosamina/metabolismo , Proteínas Ligadas a GPI/metabolismo , Lectinas/metabolismo , Mesotelioma/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Cromatografía Liquida/métodos , Células Epitelioides/metabolismo , Glicosilación , Humanos , Espectrometría de Masas/métodos , Mesotelina , Mesotelioma Maligno/metabolismo , Análisis por Matrices de Proteínas/métodosRESUMEN
INTRODUCTION: Molecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients. METHODS: Thirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed. RESULTS: Five patients relapsed during the follow-up (range 37-123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients. CONCLUSIONS: TNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools.
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Polisacáridos/metabolismo , Análisis de Matrices Tisulares , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/cirugía , Susceptibilidad a Enfermedades , Humanos , Pronóstico , Recurrencia , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND/AIM: Triple-negative breast cancer (TNBC) remains difficult to treat and new molecular targets are needed. Here, we investigated the impact of glycosyltransferase genes on TNBC patient survival. PATIENTS AND METHODS: mRNA expression levels of 101 glycosyltransferase genes in TNBC patients were compared for correlation with patient survival using The Cancer Genome Atlas data. An antibody to ß-3-N-acetylgluco-saminyltransferase 8 (B3GNT8) was applied to investigate B3GNT8 protein distribution and expression levels in 23 TNBC surgical specimens. RESULTS: B3GNT8 mRNA levels inversely correlated with relapse-free survival (p<0.01) and overall survival (p<0.05) in TNBC patients. Anti-B3GNT8 antibody binding was observed as dots in the cytoplasm of cancer cells. These dots were supposed to correspond to B3GNT8 protein in tumour cells, but their number was smaller in relapsed patients than in non-relapsed patients. CONCLUSION: B3GNT8 mRNA expression levels in TNBC tumour tissues are potentially useful in distinguishing patients with favourable and poor clinical outcomes.
Asunto(s)
Citoplasma/metabolismo , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Recurrencia , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Growth of colon carcinoma cells transfected with mucin 1 (MUC1) was effectively suppressed by vaccination with MUC1 cDNA. The suppression was dependent on the presence of Fas ligand (FasL) in the cecum, whereas it was independent of FasL in the spleen and in the liver, as revealed by the use of gld/gld mice as the recipients of vaccination, and transplantation of tumor cells expressing MUC1. CD4(+) T cells were transferred from mice immunized with MUC1 cDNA to naive gld/gld or C57BL/6 mice, and the suppression of colon carcinoma growth in the cecum was tested. The results clearly showed that FasL in the recipient played a significant role. In the cecum, FasL was associated with intratumoral CD11b(+) cells, which are likely to be responsible for vaccine-induced tumor suppression. The T cell response to MUC1 was not influenced by the gld/gld status.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/inmunología , Proteína Ligando Fas/inmunología , Mucina-1/inmunología , Neoplasias Experimentales/inmunología , Animales , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Ciego/inmunología , Ciego/metabolismo , Línea Celular Tumoral , ADN Complementario/genética , Proteína Ligando Fas/metabolismo , Humanos , Inmunización/métodos , Hígado/inmunología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucina-1/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Bazo/inmunología , Bazo/metabolismo , Carga TumoralRESUMEN
Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1(-/-) mice) and their wild-type littermates. Mgl1(-/-) mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1(-/-) mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1(-/-) mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.