Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells.
(49/2610)
Molecular chaperones are presumed to associate with large secretory mucin glycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involvement of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase labelling of MUC2 and MUC5AC with [(35)S]methionine/cysteine ([(35)S]Promix) was performed using LS180 and HT29/A1 colonic carcinoma cell lines and was followed by immunoprecipitation with anti-mucin and anti-chaperone antibodies. The precipitated labelled mucin precursors were analysed by SDS/PAGE and autoradiography. Using antibodies specific for each mucin, newly synthesized monomeric precursors of both MUC2 and MUC5AC were detected after a 15 min pulse and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using anti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2 h chase. At this time, MUC2 immunoprecipitated by anti-MUC2 was completely oligomerized and was endo-beta-N-acetylglucosaminidase-resistant, indicating that the mucin had reached the Golgi region. MUC2 co-precipitated with CRT at zero time and 0.5 h was endo-beta-N-acetylglucosaminidase-sensitive; therefore CRT must have associated with MUC2 in the ER. Treatment with tunicamycin (TUN) diminished the binding of MUC2 to CRT, suggesting a requirement for initial N-glycan addition during this process. Using anti-CLN, only a weak co-precipitation of MUC2, compared with that seen with anti-CRT, was detected in LS180 cells. In contrast with the findings for MUC2, there was no co-precipitation of MUC5AC with CRT or CLN from either cell line at the various time points. In conclusion, CRT and CLN appear to be involved in MUC2 synthesis at the stage of folding and oligomerization in the ER. Since no interaction of the chaperones with MUC5AC was detected at a similar stage of synthesis, these two structurally similar secretory mucins seem to have different chaperone requirements in the ER. (+info)
Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.
(50/2610)
Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins. (+info)
Distinct selectin ligands on colon carcinoma mucins can mediate pathological interactions among platelets, leukocytes, and endothelium.
(51/2610)
Selectins are adhesion molecules that mediate calcium-dependent cell-cell interactions among leukocytes, platelets, and endothelial cells. The naturally occurring vascular ligands for the selectins are mostly mucin-type glycoproteins. Increased expression and altered glycosylation of mucins are known to be prominent features of carcinoma progression. We have previously shown that all three selectins bind to colon carcinoma cell lines in a calcium-dependent fashion and that carcinoma growth and metastasis formation are attenuated in P-selectin-deficient mice. Here we show that the three recombinant soluble selectins recognize ligands within primary colon carcinoma tissue samples. Affinity chromatography showed that the ligands for all three selectins are O-sialoglycoprotease-sensitive mucins that are recognized in a calcium- and sialic acid-dependent manner. Furthermore, there are separate binding sites on the mucins for each selectin, allowing cross-binding of a single mucin molecule by more than one selectin. We also show that the selectin ligands on purified carcinoma mucins can mediate at least four different pathological interactions among platelets, leukocytes, and endothelial cells. These findings could explain some of the adhesive events of blood-borne tumor cells reported to occur with leukocytes, platelets, and endothelial cells, which are believed to play a part in modulating some early events in tumor metastases. (+info)
A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.
(52/2610)
L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing. (+info)
Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium.
(53/2610)
PURPOSE: To determine site and time of initiation of expression of the membrane-spanning mucin ASGP (rMuc4) and the goblet cell-specific, gel-forming mucin rMuc5AC by the developing rat ocular surface epithelium. METHODS: Newborn Sprague-Dawley rat pups were killed at 1, 7, and 14 days after birth. Adult rats (weight, 200 g) were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect ASGP mRNA using beta-actin as an internal control. Competitive RT-PCR was performed to quantitate rMuc5AC mRNA using an rMuc5AC-competitive reference standard (CRS) as an internal control. In situ hybridization was performed to localize ASGP and rMuc5AC mRNA. Goblet cells were detected by staining with periodic acid-Schiff (PAS) reagent. RESULTS: ASGP mRNA was detected by RT-PCR at 1 day after birth. Compared with beta-actin, the amount of ASGP mRNA showed a progressive increase from 1 to 14 days of postnatal development. By in situ hybridization, the expression of ASGP was first clearly detected at 14 days after birth at the lid margin, where the most stratification of epithelium was seen, and along the adjacent palpebral conjunctiva. This pattern was seen in rat eyelids that were not yet open but appeared about to open. In rat eyelids already open at 14 days after birth, ASGP mRNA was diffusely spread in the apical cell layer of both conjunctival and corneal epithelia. The expression of rMuc5AC was detected by RT-PCR in ocular surface epithelium in rat pups 1 day after birth. Quantitative RT-PCR showed a low level of rMuc5AC RNA expression in conjunctiva of 1-, 7-, and 14-day-old rats followed by a large increase in expression between 14 days and adulthood. The expression of rMuc5AC was first detected by in situ hybridization in a few goblet cells at 7 days after birth. One or two labeled cells were present in the fornical area; some were on the palpebral side of the fornix; others were present on the bulbar side. The distribution and time of appearance of rMuc5AC correlated with that of PAS staining of goblet cells. CONCLUSIONS: The developmental expression of the membrane-spanning mucin ASGP (rMuc4) and the gel-forming mucin rMuc5AC are regionally and temporally separated. Expression of the gel-forming mucin begins at the fornix at 7 days after birth and is correlated with the appearance of goblet cells, whereas, expression of the membrane-spanning mucin begins later at the lid margin at day 14. Expression of the membrane-spanning mucin correlates to eyelid opening. (+info)
Pigeon fanciers' lung: identification of disease-associated carbohydrate epitopes on pigeon intestinal mucin.
(54/2610)
Pigeon intestinal mucin, a complex high molecular weight glycoprotein, is a key antigen in the development of pigeon fanciers' lung (PFL). We have studied the specificity of antibodies to mucin in patients with PFL and asymptomatic antibody-positive individuals. Extensive papain digestion, which removes the non-glycosylated regions of the mucin leaving the heavily glycosylated 'bottle brush' regions, resulted in a 600-fold decrease in IgG3 antibody titres with little effect on IgG1 and IgG2 titres. This suggests that IgG1 and IgG2 are directed against the region rich in O-linked sugar chains whilst the majority of the IgG3 is directed against epitopes which are proteinase-sensitive. Lectin mapping of the carbohydrates present on pigeon intestinal mucin demonstrated high levels of exposed N-acetyl neuraminic acid, N-acetyl galactosamine and N-acetyl glucosamine, with lower levels of fucose and some galactose. Sera from pigeon fanciers inhibited binding of lectins specific for N-acetyl neuraminic acid, N-acetyl galactosamine, internal N-acetyl glucosamine and fucose. Sera from people with PFL, compared with sera from asymptomatic antibody-positive fanciers, had significantly higher titres of antibody that inhibited binding of four lectins specific for N-acetyl galactosamine and one fucose-specific lectin, suggesting that these sugars may play a dominant role in disease-associated epitopes. The results suggest that different IgG subclasses recognize different epitopes on mucin and that the epitopes recognized by the major subclasses are present on the O-linked oligosaccharides. Further, the carbohydrate-specific anti-mucin antibodies produced by PFL patients may differ in their specificity from those found in asymptomatic individuals. (+info)
The trefoil peptides TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response.
(55/2610)
Members of the trefoil factor (TFF) family are mucin-associated polypeptides that are expressed along the entire length of the gastrointestinal tract. TFFs have been proposed to play a role in mucosal defence through both protective and reparative mechanisms. The potential relationship between TFFs and mucins in non-gut glycoprotein-secreting epithelia has not been fully explored. In the present study we identified TFF2 and TFF3 mRNA and peptide in rat lymphoid tissues, demonstrated that TFF peptide expression in rat spleen increased 1.5- to 3-fold following experimental induction of the immune response, and showed that hTFF2 and hTFF3 (1-5 mg/ml) stimulated migration of human monocytes. Our data suggest that TFFs may in part be involved in the repair of injury through the modulation of the inflammatory response. (+info)
Expression of core 2 beta-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes.
(56/2610)
Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes. (+info)