Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis^a, as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85–120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.     

Galectin-1 binds mucin in human trophoblast

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

In vivo glycosylation of mucin tandem repeats.

The biochemical and biophysical properties of mucins are largely determined by extensive O-glycosylation of serine- and threonine-rich tandem repeat (TR) domains. In a number of human diseases aberrant O-glycosylation is associated with variations in the properties of the cell surface-associated and secreted mucins. To evaluate in vivo the O-glycosylation of mucin TR domains, we generated recombinant chimeric mucins with TR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted for the native TRs of epitope-tagged MUC1 protein (MUC1F). These hybrid mucins were extensively O-glycosylated and showed the expected association with the cell surface and release into culture media. The presence of different TR domains within the chimeric mucins appears to have limited influence on their posttranslational processing. Alterations in glycosylation were detailed by fast atom bombardment mass spectrometry and reactivity with antibodies against particular blood-group and tumor-associated carbohydrate antigens. Future applications of these chimeras will include investigations of mucin posttranslational modification in the context of disease.     

An O-acetylated sialyl-Tn is involved in ovarian cancer-associated antigenicity

We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.     

Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity

Mucins are major epithelial luminal surface proteins and function as a physical and biological barrier protecting mucous epithelia. Diverse glycosylation of mucins potentially provides a basis for tissue-specific interaction with the milieu. When mucins are associated with malignant epithelial cells, they not only protect these cells from a host environment during metastatic dissemination but also generate immunogenic epitopes which are used by the host in the detection and immunological elimination of carcinoma cells potentially depending upon their status of glycosylation. Diverse mucin structures are generated by the combination of different core peptides, of which 10 have been reported so far, multiple types of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts), and the consequences of stepwise glycosylation events. For example, the mucin 1 (MUC1) associated with malignant cells was previously believed to exhibit unique features with a lower percentage of threonine and serine residues attached to N-acetylgalactosamine and/or without extension through core 2 structures. Some of MUC1-specific monoclonal antibodies and cytotoxic lymphocytes recognize the peptide sequences PDTR within the tandem repeat portion exposed by decreased degree of glycosylation. The specific arrangement of N-acetylgalactosamine residues is shown to be generated by a combination of pp-GalNAc-Ts with different specificities. The role of core 2 branching is somewhat confusing because well-known carcinoma-associated carbohydrate epitopes such as sialyl-Le(X), sialyl-Le(a), Le(Y), and others are often expressed when O-glycans are extended through core 2 branching. The other series of well-known carcinoma-associated carbohydrate structures are truncated O-glycans, conventionally called Tn and sialyl-Tn antigens. Interestingly, these are often found to be aligned on core polypeptides, resulting in three or more consecutive truncated O-glycans. MUC2 and other mucins, but not MUC1, have unique tandem repeats containing three or more consecutive serine or threonine residues, which potentially serve as a scaffold for trimeric Tn and sialyl-Tn epitopes. We recently found, using the MUC2 tandem repeat, that trimeric Tn is a high-affinity receptor for a calcium-type lectin expressed on the surface of histiocytic macrophages. The biosynthesis of trimeric Tn was strictly regulated by the acceptor specificity of pp-GalNAc-Ts. These results strongly suggest that variation in both glycan structures and distribution of glycans on the core polypeptides give mucins unique and diverse biological functions that play essential roles in carcinoma-host and other cellular interactions.     

Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins.

Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbohydrate markers of pancreatic cancer

Pancreatic cancer is the fourth most common cause of death from cancer in the world and the sixth in Europe. Pancreatic cancer is more frequent in males than females. Worldwide, following diagnosis of pancreatic cancer, <2% of patients survive for 5 years, 8% survive for 2 years and <50% survive for only approx. 3 months. The biggest risk factor in pancreatic cancer is age, with a peak of morbidity at 65 years. Difficulty in the diagnosis of pancreatic cancer causes a delay in its detection. It is one of the most difficult cancers to diagnose and therefore to treat successfully. Additional detection of carbohydrate markers may offer a better diagnosis of pancreatic cancer. Carbohydrate markers of cancer may be produced by the cancer itself or by the body in response to cancer, whose presence in body fluids suggests the presence and growth of the cancer. The most widely used, and best-recognized, carbohydrate marker of pancreatic cancer is CA 19-9 [CA (carbohydrate antigen) 19-9]. However, the relatively non-specific nature of CA 19-9 limits its routine use in the early diagnosis of pancreatic cancer, but it may be useful in monitoring treatment of pancreatic cancer (e.g. the effectiveness of chemotherapy), as a complement to other diagnostic methods. Some other carbohydrate markers of pancreatic cancer may be considered, such as CEA (carcinoembryonic antigen), CA 50 and CA 242, and the mucins MUC1, MUC2 and MUC5AC, but enzymes involved in the processing of glycoconjugates could also be involved. Our preliminary research shows that the activity of lysosomal exoglycosidases, including HEX (N-acetyl-β-D-hexosaminidase), GAL (β-D-galactosidase), FUC (α-L-fucosidase) and MAN (α-D-mannosidase), in serum and urine may be used in the diagnosis of pancreatic cancer.     

Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.     

Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

Mucin glycoproteins in neoplasia

Mucins are high molecular weight glycoproteins that are heavily glycosylated with many oligosaccharide side chains linked O-glycosidically to the protein backbone. With the recent application of molecular biological methods, the structures of apomucins and regulation of mucin genes are beginning to be understood. At least nine human mucin genes have been identified to date. Although a complete protein sequence is known for only three human mucins (MUC1, MUC2, and MUC7), common motifs have been identified in many mucins. The pattern of tissue and cell-specific expression of these mucin genes are emerging, suggesting a distinct role for each member of this diverse mucin gene family. In epithelial cancers, many of the phenotypic markers for pre-malignant and malignant cells have been found on the carbohydrate and peptide moieties of mucin glycoproteins. The expression of carbohydrate antigens appears to be due to modification of peripheral carbohydrate structures and the exposure of inner core region carbohydrates. The expression of some of the sialylated carbohydrate antigens appears to correlate with poor prognosis and increased metastatic potential in some cancers. The exposure of peptide backbone structures of mucin glycoproteins in malignancies appears to be due to abnormal glycosylation during biosynthesis. Dysregulation of tissue and cell-specific expression of mucin genes also occurs in epithelial cancers. At present, the role of mucin glycoproteins in various stages of epithelial cell carcinogenesis (including the preneoplastic state and metastasis), in cancer diagnosis and immunotherapy is under investigation.     

Characterization of mucins in human middle ear and Eustachian tube

Mucins are important glycoproteins in the mucociliary transport system of the middle ear and Eustachian tube. Little is known about mucin expression within this system under physiological and pathological conditions. This study demonstrated the expression of MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas only MUC5B mucin expression was demonstrated in noninflamed middle ears. MUC5B and MUC4 mucin genes were upregulated 4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes was accompanied by an increase of MUC5B- and MUC4-producing cells in the middle ear mucosa. Electron microscopy of the secretions from COM and MOM showed the presence of chainlike polymeric mucin. These data indicate that the epithelium of the middle ear and Eustachian tube expresses distinct mucin profiles and that MUC5B and MUC4 mucins are highly produced and secreted in the diseased middle ear. These mucins may form thick mucous effusion in the middle ear cavity and compromise the function of the middle ear.     

CFTR expression does not influence glycosylation of an epitope-tagged MUC1 mucin in colon carcinoma cell lines

The cause of the mucus clearance problems associated with cystic fibrosis remains poorly understood though it has been suggested that mucin hypersecretion, dehydration of mucins, and biochemical abnormalities in the glycosylation of mucins may be responsible. Since the biochemical and biophysical properties of a mucin are dependent on O-glycosylation, our aim was to evaluate the O-glycosylation of a single mucin gene product in matched pairs of cells that differed with respect to CFTR expression. An epitope-tagged MUC1 mucin cDNA (MUC1F) was used to detect variation in mucin glycosylation in stably transfected colon carcinoma cell lines HT29 and Caco2. The glycosylation of MUC1F mucin was evaluated in matched pairs of Caco2 cell lines that either express wild-type CFTR or have spontaneously lost CFTR expression. The general glycosylation pattern of MUC1F was evaluated by determining its reactivity with a series of monoclonal antibodies against known blood group and tumor-associated carbohydrate antigens. Metabolic labeling experiments were used to estimate the gross levels of glycosylation and sulfation of MUC1F mucin in these matched pairs of cell lines. Expression of CFTR in this experimental system did not affect the gross levels of glycosylation or sulfation of the MUC1F mucin nor the types of carbohydrates structures attached to the MUC1F protein.     

Human biliary mucin binds to E-selectin: a possible role in modulation of inflammation

E-selectin, expressed on endothelial cells, mediates adhesion of leukocytes and tumor cells to endothelium. CA19-9 (sialyl-Lewis(a)) and sialyl-Lewis(x) are specific ligands for E-selectin. We have recently shown that mucin-rich culture media from human gallbladder epithelial cells contains CA19-9. In this study, we have tested whether human biliary mucin binds to E-selectin. The ability of mucins to inhibit the adhesion of HL-60 cells to immobilized E-selectin was taken as an index for E-selectin binding. Gallbladder bile, hepatic bile, and culture medium from human gallbladder epithelial cells completely inhibited the adhesion of HL-60 cells to E-selectin. The mucin-rich fractions of human bile exhibited strong inhibition, whereas mucin-free fractions had little effect. In contrast to human bile samples, CA19-9-free medium from cultured dog gallbladder epithelial cells failed to inhibit HL-60 binding. Furthermore, after CA19-9 immunoaffinity chromatography, which selectively extracted CA19-9 from bile, bile samples showed poor inhibition of HL-60 adhesion to immobilized E-selectin. A good correlation was observed between E-selectin binding and CA 19-9 concentrations in bile. Our results show that human bile has E-selectin binding activity that is mediated by the CA19-9 side chain of biliary mucin.     

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293-302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155-165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.     

2010 Reviewers for Biotechnic & Histochemistry

Pancreatic cancer is characterized by aggressive growth and resistance to treatment. Identification of unique biomarkers for diagnosis and prognosis is important for treatment of this disease. We investigated the expression patterns of mucin 1 (MUC1), mucin 2 (MUC2) and cytokeratin 17 (CK17) in both normal tissues and metastatic adenocarcinomas using immunohistochemistry (IHC). We have shown that MUC1 (pan-epithelial membrane mucin), MUC2 (intestinal-type secretory mucin) and CK17 can be used as a panel of markers to distinguish collectively pancreatobiliary carcinoma from other primary site carcinomas. Tumors originating in the pancreatobiliary system showed an expression pattern of MUC1 (+), MUC2 (?) and CK17 (+). By contrast, tumors arising from the colorectal region were MUC1 (?), MUC2 (+) and CK17 (?), while tumors originating from non-pancreatobiliary system tissue expressed a MUC1 (+), MUC2 (?) and CK17 (?) profile. More importantly, the MUC1 (+), MUC2 (?) and CK17 (+) result showed greater sensitivity than CA19-9 by IHC, which is the currently accepted and widely used pancreatic tumor marker for diagnosing pancreatic cancer. Thirteen of 51 cases (25%) of pancreatobiliary adenocarcinomas with the pattern MUC1 (+), MUC2 (?) and CK17 (+) showed no immunoreactivity for CA19-9, while 34/51 (67%) cases having MUC1 (+), MUC2 (?) and CK17 (+) were correlated with positive CA19-9 staining. Our data support using an antibody panel of MUC1, MUC2 and CK17 to enhance current methods for pancreatic cancer diagnosis by identifying specifically the primary tissue of origin.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.