Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.

Localization of transmembrane mucin MUC1 on the apical surface of oral mucosal cells.

The purpose of the present study was to demonstrate the localization of transmembrane mucin MUC1 on the outer layer of oral mucosal cells and the involvement of apical cell surface microplicae (MPL) in bioadhesion of MUC1. Tissue samples of six healthy subjects were obtained. First, the presence of MUC1 was examined with an immunohistochemical method using a monoclonal MUC1 antigen called HFMG1. Second, the localization of MUC1 was examined with immuno-scanning electron microscopy. Immunohistochemically, high intense staining for MUC1 (antigen HFMG1) was detected in the epithelial superficial layers. In the superficial layer, intense MUC1 expression was seen predominantly on the apical cell surface. On the apical epithelial cells, MUC1 was associated predominantly with MPL towards the oral cavity. The novelty of the results of the present study is that MPL serves a harbor of MUC1 in superficial epithelial cells towards the oral cavity. It is speculated that the transmembrane MUC1 is one component of the "oral mucosal barrier complex" representing a signaling pathway between saliva and mucosal cells.Abbreviations: MUC1: mucin1; MAM: membrane-anchored mucin; OMBC: oral mucosal barrier complex; LM: light microscopy; TEM: transmission electron microscopy; SEM: scanning electron microscopy; iSEM: immuno-scanning electron microscopy; MPL: microplicae.

Preferential Localization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small Cell Lung Carcinoma Cells.

Lung cancer remains to be the leading cause of cancer-related mortality worldwide. Finding new noninvasive biomarkers for lung cancer is still a significant clinical challenge. Exosomes are membrane-bound, nano-sized vesicles that are released by various living cells. Studies on exosomal proteomics may provide clues for developing clinical assays. In this study, we performed semi-quantitative proteomic analysis of proteins that were purified from exosomes of NCI-H838 non-small cell lung cancer cell line, with total cellular membrane proteins as control. In the exosomes, LC-MS/MS by data-independent analysis mode identified 3235 proteins. THBS1, ANXA6, HIST1H4A, COL18A1, MDK, SRGN, ENO1, TUBA4A, SLC3A2, GPI, MIF, MUC1, TALDO1, SLC7A5, ICAM1, HSP90AA1, G6PD, and LRP1 were found to be expressed in exosomes at more than 5-fold higher level as compared to total cellular membrane proteins. A well-known cancer biomarker, MUC1, is expressed at 8.98-fold higher in exosomes than total cellular membrane proteins. Subsequent analysis of plasma exosomes from non-small cell lung cancer (NSCLC) patients by a commercial electrochemiluminescence immunoassay showed that exosomal MUC1 level is 1.5-fold higher than healthy individuals (mean value 1.55 ± 0.16 versus mean value 1.05 ± 0.06, p = 0.0213). In contrast, no significant difference of MUC1 level was found between NSCLC patients and healthy individuals' plasma (mean value 5.48 ± 0.65 versus mean value 4.16 ± 0.49). These results suggest that certain proteins, such as MUC1, are selectively enriched in the exosome compartment. The mechanisms for their preferential localization and their biological roles remain to be studied.

Immunohistochemical and immunoelectron microscopic studies of the localization of KL-6 and epithelial membrane antigen (EMA) in presumably normal pulmonary tissue and in interstitial pneumonia.

To clarify the localization of KL-6 and epithelial membrane antigen (EMA) in human lungs, immune reactions to antibodies to these factors were examined in detail at light and electron microscopic levels. Immunohistochemical investigation was performed in 17 cases of usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP), hypersensitivity pneumonitis (HP), collagen vascular disease-associated interstitial pneumonias (CVD-IP), viral pneumonia, and bronchobronchioloectasis, as well as in 10 cases of presumably normal pulmonary tissue resected as a result of spontaneous pneumothorax. Immunohistochemical study revealed similar discontinuous linear or dome-shaped positive patterns restricted to type II alveolar cells in presumably normal tissue and only some regions of interstitial pneumonia. In sharp contrast, immune reactions with each of the two antibodies yielded a continuous linear pattern surrounding damaged areas in most regions of interstitial pneumonias and some normal areas as well. Staining for EMA antibody was negative in some regenerating alveolar and bronchial cells in regenerating foci in interstitial pneumonias, although staining for KL-6 antibody was always positive in these cells. Immunoelectron microscopic studies demonstrated similar positive reactions with both antibodies on the surface of alveolar epithelial cells in three of the cases examined, with surface positive granules 100-200 nm in diameter. Thus, although staining for both KL-6 and EMA antibodies exhibited discontinuous positivity restricted to type II alveolar cells in nondamaged regions, immune reactions were continuous and linear in pattern in or around damaged areas of the lungs at both light and electron microscopic levels, probably as a consequence of cell-surface barrier function. These findings in pulmonary tissue might be evidence of defense functions.

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells.

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.