RESUMO
This review describes the organization and importance of mucus in the intestine and lungs in relation to the diseases cystic fibrosis (CF), ulcerative colitis and chronic obstructive pulmonary disease (COPD). The inner surfaces of the body are protected by mucus built around polymeric glycoproteins called mucins. In the disease CF, the small intestinal mucus is in contrast the normal attached to the epithelium, explaining the intestinal problems at this disease. The inner of the two mucus layers of colon is normally impenetrable to bacteria, keeping the commensals away from and protecting the epithelium. This impenetrable property is dependent on the bacterial composition and the host diet, observations that can explain the increased incidence of inflammatory bowel diseases in the western world as bacteria reach the epithelial cells in active ulcerative colitis. The respiratory tract is normally cleared by thick mucus bundles that moved by the cilia sweep the epithelial surface. In CF, the bundles are nonmoving already at birth. Cholinergic stimulations stop the bundle movement explaining some of the beneficial effect of anticholinergic treatment in COPD. In this disease as well as in more developed CF, an attached mucus layer is formed. This mucus has features similar to the protective inner colon mucus and is by this able to separate bacteria from the epithelial surface. When formed in healthy individuals this mucus can be coughed up, but in chronically diseased lungs, bacteria colonizing the mucus will remain in the lungs and the resulting inflammation contribute to the destruction of the lungs.
Assuntos
Colite Ulcerativa , Fibrose Cística , Mucinas/fisiologia , Muco/fisiologia , Doença Pulmonar Obstrutiva Crônica , Colite Ulcerativa/fisiopatologia , Fibrose Cística/fisiopatologia , Humanos , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
BACKGROUND: Anastomotic leakage (AL) is the most dreaded complication after colorectal surgery, causing high morbidity and mortality. Mucus is a first line of defence against external factors in the gastrointestinal tract. In this study, the structural mucus protein Muc2 was depleted in genetically engineered mice and the effect on healing of colonic anastomoses studied in an experimental model. METHODS: Mice of different Muc2 genotypes were used in a proximal colonic AL model. Tissues were scored histologically for inflammation, bacterial translocation was determined by quantitative PCR of bacterial 16S ribosomal DNA, and epithelial cell damage was determined by assessing serum levels of intestinal fatty acid-binding protein. RESULTS: Of 22 Muc2-deficient (Muc2-/- ) mice, 20 developed AL, compared with seven of 22 control animals (P < 0·001). Control mice showed normal healing, whereas Muc2-/- mice had more inflammation with less collagen deposition and neoangiogenesis. A tendency towards higher bacterial translocation was seen in mesenteric lymph nodes and spleen in Muc2-/- mice. Intestinal fatty acid-binding protein levels were significantly higher in Muc2-/- mice compared with controls (P = 0·011). CONCLUSION: A functional mucous layer facilitates the healing of colonic anastomoses. Clinical relevance Colorectal anastomotic leakage remains the most dreaded complication after colorectal surgery. It is known that the aetiology of anastomotic leakage is multifactorial, and a role is suggested for the interaction between intraluminal content and mucosa. In this murine model of proximal colonic anastomotic leakage, the authors investigated the mucous layer at the intestinal mucosa, as the first line of defence, and found that a normal, functioning mucous layer is essential in the healing process of colonic anastomoses. Further research on anastomotic healing should focus on positively influencing the mucous layer to promote better postoperative recovery.
Assuntos
Anastomose Cirúrgica , Cirurgia Colorretal , Cicatrização/fisiologia , Fístula Anastomótica/prevenção & controle , Animais , Translocação Bacteriana , Colo/cirurgia , Dinoprostona/farmacologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Ligação a Ácido Graxo/sangue , Genótipo , Mucosa Intestinal , Camundongos , Modelos Teóricos , Mucina-2/genética , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/genéticaRESUMO
AIM: Downregulated in adenoma (DRA, Slc26a3) is a member of the solute carrier family 26 (SLC26), family of anion transporters, which is mutated in familial chloride-losing diarrhoea (CLD). Besides Cl(-) -rich diarrhoea, CLD patients also have a higher-than-average incidence of intestinal inflammation. In a search for potential explanations for this clinical finding, we investigated colonic electrolyte transport, the mucus layer and susceptibility against dextran sodium sulphate (DSS)-induced colitis in Slc26a3(-/-) mice. METHODS: HCO3 (-) secretory (JHCO3 (-) ) and fluid absorptive rates were measured by single-pass perfusion in vivo and in isolated mid-distal colonic mucosa in Ussing chambers in vitro. Colonocyte intracellular pH (pHi ) was assessed fluorometrically, the mucus layer by immunohistochemistry and colitis susceptibility by the addition of DSS to the drinking water. RESULTS: HCO3 (-) secretory (JHCO3- ) and fluid absorptive rates were strongly reduced in Slc26a3(-/-) mice compared to wild-type (WT) littermates. Despite an increase in sodium/hydrogen exchanger 3 (NHE3) mRNA and protein expression, and intact acid-activation of NHE3, the high colonocyte pH in Slc26a3(-/-) mice prevented Na(+) /H(+) exchange-mediated fluid absorption in vivo. Mucin 2 (MUC2) immunohistochemistry revealed the absence of a firm mucus layer, implying that alkaline secretion and/or an absorptive flux may be necessary for optimal mucus gel formation. Slc26a3(-/-) mice were highly susceptible to DSS damage. CONCLUSIONS: Deletion of DRA results in severely reduced colonic HCO3 (-) secretory rate, a loss of colonic fluid absorption, a lack of a firmly adherent mucus layer and a severely reduced colonic mucosal resistance to DSS damage. These data provide potential pathophysiological explanations for the increased susceptibility of CLD patients to intestinal inflammation.
Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Acidose/genética , Acidose/metabolismo , Animais , Antiporters/genética , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mucina-2/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de SulfatoRESUMO
CD43 is a highly glycosylated transmembrane protein expressed on the surface of most hematopoietic cells. Expression of CD43 has also been demonstrated in many human tumor tissues, including colon adenomas and carcinomas, but not in normal colon epithelium. The potential contribution of CD43 to tumor development is still not understood. Here, we show that overexpression of CD43 increases cell growth and colony formation in mouse and human cells lacking expression of either p53 or ARF (alternative reading frame) tumor-suppressor proteins. In addition, CD43 overexpression also lowers the detection of the FAS death receptor on the cell surface of human cancer cells, and thereby helps to evade FAS-mediated apoptosis. However, when both p53 and ARF proteins are present, CD43 overexpression activates p53 and suppresses colony formation due to induction of apoptosis. These observations suggest CD43 as a potential contributor to tumor development and the functional ARF-p53 pathway is required for the elimination of cells with aberrant CD43 expression.
Assuntos
Apoptose/fisiologia , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Genes p53 , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Leucossialina/fisiologia , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/deficiência , Receptor fas/fisiologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucossialina/genética , Camundongos , Neoplasias/patologia , Transdução de Sinais/genéticaRESUMO
The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.
Assuntos
Reatores Biológicos , Biotecnologia/métodos , Células CHO , Mucina-1/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Contagem de Células , Técnicas de Cultura de Células , Linhagem Celular , Cricetinae , Meios de Cultura Livres de Soro , Expressão Gênica , Glucose/farmacologia , Glutamina/farmacologia , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Mucina-1/genética , Fatores de TempoRESUMO
CD43 is an abundant transmembrane sialoglycoprotein in leukocyte-type cell lines, but it has also been suggested to be present in colon adenomas and colon carcinomas. We have now shown that CD43 is expressed in a variety of cell lines of different origins (CaSKI, A549, 293, MTSV1-7, MCF7, HT-1080, Jurkat, K562, COLO 205, HT-29, Caco-2, DLD-1 and SW480). The level of expression of CD43 mRNA was analyzed by reverse transcriptase-polymerase chain reaction and that of the protein by immunoprecipitation and Western blot, flow cytometry and confocal microscopy using two monoclonal anti-CD43 antibodies (L10 and 4D2). As all cell lines expressed CD43, it is suggested that CD43 has a more fundamental function than previously believed and thus cannot be considered only as a specific leukocyte marker.
Assuntos
Antígenos CD , Sialoglicoproteínas/biossíntese , Anticorpos Monoclonais/metabolismo , Western Blotting , Linhagem Celular , Separação Celular , Eletroforese em Gel de Poliacrilamida , Éxons , Citometria de Fluxo , Humanos , Íntrons , Células Jurkat , Leucossialina , Microscopia Confocal , Microscopia de Fluorescência , Plasmídeos/metabolismo , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/metabolismo , Células Tumorais CultivadasRESUMO
Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.
Assuntos
Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Macrolídeos , Mucinas/metabolismo , Cloreto de Amônio/farmacologia , Antibacterianos/farmacologia , Compartimento Celular/efeitos dos fármacos , Endossomos/química , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/enzimologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.
Assuntos
Adenocarcinoma/imunologia , Antígenos CD , Neoplasias do Colo/imunologia , Selectina E/metabolismo , Epitopos/metabolismo , Gangliosídeos/imunologia , Mucina-1/metabolismo , Oligossacarídeos/imunologia , Sialoglicoproteínas/metabolismo , Adenocarcinoma/metabolismo , Animais , Antígeno CA-19-9 , Células CHO/metabolismo , Sequência de Carboidratos , Adesão Celular/fisiologia , Neoplasias do Colo/metabolismo , Cricetinae , Selectina E/genética , Glicosilação , Células HL-60/patologia , Humanos , Leucossialina , Dados de Sequência Molecular , Mucina-1/imunologia , Coelhos , Sialoglicoproteínas/imunologia , Antígeno Sialil Lewis X , Estresse Mecânico , Células Tumorais CultivadasRESUMO
CD43 is a leukocyte-associated sialoglycoprotein which is also expressed in human colon adenoma and carcinoma. To obtain monoclonal antibodies (MAbs) that would react with CD43 in a glycosylation-independent way, antibodies were raised against a peptide corresponding to a portion of the CD43 cytoplasmic domain. Hybridomas were screened on paraffin sections from CD43-positive colon tumours. The reactivity of the antibodies with CD43 was verified by Western blot analysis of lysate of CHO cells transfected with human CD43 cDNA and by immunoprecipitation of lysates from CD43+ cell lines. Epitope mapping of antibodies was done using overlapping heptameric peptides. A detailed characterisation of one of the novel antibodies (CD43-3A1) is presented. This antibody reacts with the CD43 protein regardless of its glycosylation in Western blot analysis, immunoprecipitation and immuno-histochemistry of paraffin sections. Immuno-histochemical analysis of paraffin sections from colon adenoma and carcinoma tissues as well as colon cancer cell lines revealed that CD43 was predominantly localised intracellularly, in contrast to leukocyte-type cells. The MAb reacted more efficiently with paraffin-embedded colon adenoma and carcinoma cells than previously characterised CD43-specific antibodies. This should facilitate the evaluation of a potential role of CD43 during cancer development.
Assuntos
Adenocarcinoma/química , Adenoma/química , Anticorpos Monoclonais/imunologia , Antígenos CD , Neoplasias do Colo/química , Sialoglicoproteínas/análise , Sequência de Aminoácidos , Animais , Western Blotting , Células CHO , Cricetinae , Mapeamento de Epitopos , Humanos , Imuno-Histoquímica , Leucossialina , Dados de Sequência Molecular , Testes de Precipitina , Sialoglicoproteínas/imunologiaRESUMO
The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1-and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.
Assuntos
Imunossupressores/farmacologia , Mucina-1/imunologia , Células 3T3/imunologia , Células 3T3/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Células HL-60/imunologia , Células HL-60/metabolismo , Células HeLa , Humanos , Imunossupressores/isolamento & purificação , Imunossupressores/urina , Células Jurkat/imunologia , Células Jurkat/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mucina-1/isolamento & purificação , Mucina-1/urina , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
The O-linked oligosaccharides from three fractions of highly glycosylated mucin glycopeptides obtained from sputum of a patient with cystic fibrosis were characterized and compared regarding size, composition, sequence and when possible linkage positions. Neutral and sialic acid-containing glycans were permethylated and analyzed by high-temperature GC-MS and MALDI-MS, showing more than 60 different oligosaccharides with a size of up to 15 monosaccharide units. Some of the observed oligosaccharides are novel for respiratory secretions, one being a trifucosylated heptasaccharide with the proposed structure: Fuc-Gal-4(Fuc-3)GlcNAc-(Fuc-)Gal-3GalNAcol. The glycosylation of two of the glycopeptide fractions was similar with regard to the neutral and sialylated oligosaccharides despite their different origins from the sol or gel phase. Analysis of the sulfated oligosaccharides by FAB-MS/MS indicated that the gel fraction contained C-6 linked sulfate groups while the two sol fractions also contained C-3 linked sulfate. The results suggest the presence of different glycosylated mucin domains, probably originating from different mucin glycoforms and/or apoproteins in the airway of cystic fibrosis patients.
Assuntos
Fibrose Cística , Glicopeptídeos/química , Mucinas/química , Escarro/química , Adolescente , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Glicosilação , Humanos , Dados de Sequência Molecular , Oligossacarídeos/química , Análise de Sequência , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ésteres do Ácido SulfúricoRESUMO
Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse-chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.
Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Mucinas/química , Mucinas/metabolismo , Sequência de Aminoácidos , Animais , Bioquímica/métodos , Carcinoma/química , Carcinoma/metabolismo , Neoplasias do Colo/química , Neoplasias do Colo/metabolismo , Reações Cruzadas , Dimerização , Mucinas Gástricas/imunologia , Mucinas Gástricas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Soros Imunes , Dados de Sequência Molecular , Mucina-5AC , Mucina-2 , Mucinas/imunologia , Coelhos , Frações Subcelulares , Células Tumorais Cultivadas , Tunicamicina/farmacologia , UltracentrifugaçãoRESUMO
Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Antibacterianos/farmacologia , Transporte Biológico Ativo , Biomarcadores Tumorais , Dimerização , Retículo Endoplasmático/efeitos dos fármacos , Mucinas Gástricas/biossíntese , Glicosilação , Complexo de Golgi/efeitos dos fármacos , Humanos , Mucina-2 , Frações Subcelulares/metabolismo , Células Tumorais Cultivadas , Tunicamicina/farmacologia , UltracentrifugaçãoRESUMO
The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.
Assuntos
Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Dimerização , Glicosilação , Guanidina , Humanos , Concentração de Íons de Hidrogênio , Mucina-2 , Conformação Proteica , Solubilidade , Células Tumorais Cultivadas , Ultracentrifugação , ÁguaRESUMO
The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with core proteins of the heavily glycosylated MUC1 mucins from the colon carcinoma cell line COLO205 and bile. Both these mucins (designated as H-CanAg and SBG2) are highly glycosylated having 15 and 50 sugar residues per oligosaccharide, respectively. Only a few of the antibodies (129, 139, 153 and 162) reacted with both SBG2 and H-CanAg, and not with a control mucin (L-CanAg) having a similar glycosylation as H-CanAg. These antibodies were tested for their ability to catch soluble mucins, and the antibody 162 was found to be good also in this type of assay. The antibodies selected here should be useful for the detection of high glycosylated forms of the MUC1 mucin in tissues and serum.
Assuntos
Anticorpos Monoclonais/análise , Bile/imunologia , Neoplasias do Colo/imunologia , Mucina-1/imunologia , Fluorimunoensaio , Glicosilação , Humanos , Oligossacarídeos , Células Tumorais CultivadasRESUMO
The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcalpha1-3(GalNAcbeta1-4)Galbeta1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcalpha1-3Galbeta1- were found in all samples. Core 1 (Galbeta1-3GalNAcalpha1-), core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-), core 3 (GlcNAcbeta1-3GalNAcalpha1-), and core 4 (GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc.
Assuntos
Mucosa Intestinal/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Mucinas/química , Mucinas/metabolismo , Oligossacarídeos/química , Sistema ABO de Grupos Sanguíneos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia em Gel , Epitopos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Glicosilação , Mucosa Intestinal/química , Dados de Sequência Molecular , Mucina-2 , Mucinas/biossíntese , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Oligossacarídeos/isolamento & purificação , Especificidade de Órgãos , Ratos , Ácidos Siálicos/análiseRESUMO
CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we report the expression of CD43 in the epithelial cells of all studied colorectal adenomas (21/21) and in about 50% (18/34) of adenocarcinomas as analyzed both at the mRNA and protein levels. Direct evidence showing the causal role of CD43 in colon tumorigenesis is lacking, but its involvement in leukocyte activation and impairment of apoptotic response suggests a role for CD43 in colon cancer development.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Antígenos CD , Neoplasias Colorretais/metabolismo , Sialoglicoproteínas/biossíntese , Humanos , Imuno-Histoquímica , Hibridização In Situ , LeucossialinaRESUMO
The effect of two secreted mucin-type glycoproteins on natural killer (NK) cell cytotoxicity against K562 target cells has been studied. These mucins carry the carcinoma-associated sialyl-Lewis a carbohydrate epitopes and were purified from the human colon adenocarcinoma cell line COLO 205 secretions, where they lack their cytoplasmic parts. The larger one has an apoprotein encoded by the MUC1 gene, and the smaller one has CD43 (leukosialin) as the core protein. The purified MUC1 mucin could inhibit the target cell lysis by NK cells in a dose-response-dependent way, whereas other mucin domains of similar size showed no inhibition. The second mucin, CD43, inhibited lysis by NK cells, although less than the larger one. The MUC1 mucin bound to the enriched natural killer cell preparations in a partial Ca2+-dependent way as well. This mucin also bound to the target cells. The K562 cells, normally expressing high amount of CD43, showed an increased resistance to lysis by NK cells when transfected with MUC1 cDNA compared with nontransfected cells. One can speculate that mucins secreted or expressed in the plasma membrane of cancer cells could interfere with NK cell-mediated lysis.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Mucina-1/metabolismo , Mucina-1/fisiologia , Adenocarcinoma/imunologia , Antígenos CD/biossíntese , Antígenos CD/isolamento & purificação , Antígenos CD/farmacologia , Ligação Competitiva/imunologia , Neoplasias do Colo/imunologia , Testes Imunológicos de Citotoxicidade , Humanos , Células Matadoras Naturais/imunologia , Leucossialina , Mucina-1/isolamento & purificação , Sialoglicoproteínas/biossíntese , Sialoglicoproteínas/isolamento & purificação , Sialoglicoproteínas/farmacologia , Células Tumorais CultivadasRESUMO
The colon carcinoma cell line COLO 205 has earlier been shown to express and secrete two mucin-type glycoproteins, the leukocyte-associated sialoglycoprotein CD43 or leukosialin (named L-CanAg) and the MUC1 mucin (named H-CanAg). Both glycoproteins carry sialyl-Lewis a epitopes and could bind transfected COS cells expressing E-selectin in a Ca(2+)- and E-selectin-dependent way. Using the monoclonal antibodies C50, C241 (both against sialyl-Lewis a), and CSLEX1 (against sialyl-Lewis x), the MUC1 mucin was shown to express both sialyl-Lewis a and sialyl-Lewis x epitopes, while the CD43 mucin expressed sialyl-Lewis a and almost no sialyl-Lewis x epitopes. These two secreted glycoproteins could inhibit human polymorphonuclear leukocyte or HL-60 cell adhesion to E-selectin-transfected COS cells or IL-1 beta-stimulated human endothelial cells in vitro. The inhibitory efficiency of the MUC1 mucin was 5-10 times larger than that of the CD43 mucin, when studied on endothelial cells and comparable amounts of sample were used. Removing the sialic acids from the MUC1 or CD43 mucins by sialidase treatment abolished the inhibitory effect. Monoclonal antibodies against sialyl-Lewis a greatly and equally inhibited the binding of the MUC1 or CD43 mucins, whereas an antibody against sialyl-Lewis x (CSLEX1) showed almost no inhibitory effect. The result proposes that the sialyl-Lewis a epitope on at least some mucin-type molecules bind E-selectin better than sialyl-Lewis x and that the potency of tumor-secreted mucins to interfere with leukocyte attachment to E-selectin could be dependent on the apoprotein size or its presentation of the carbohydrate epitopes.
Assuntos
Antígenos CD , Neoplasias do Colo/metabolismo , Selectina E/metabolismo , Gangliosídeos/metabolismo , Mucina-1/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Anticorpos Monoclonais , Antígeno CA-19-9 , Células COS , Adesão Celular , Neoplasias do Colo/patologia , Epitopos , Gangliosídeos/imunologia , Humanos , Leucócitos/metabolismo , Leucossialina , Mucina-1/química , Oligossacarídeos/imunologia , Oligossacarídeos/metabolismo , Sialoglicoproteínas/química , Antígeno Sialil Lewis X , Células Tumorais CultivadasRESUMO
RNA from four colorectal carcinoma cell lines was prepared and analysed in Northern blots using probes for the MUC2, MUC4, and MUC5AC mucin apoprotein genes. The sizes of the transcripts were very large, in the order of at least 12-16 kb. The presence of distinct bands is in contrast to earlier reports, where these transcripts showed extensive polydispersity. RNA from rat small intestine was also prepared and probed with cDNA for the rat Muc2 mucin gene. This analysis also showed a large and discrete hybridizing band, indicating that apomucin mRNA of well-defined size can be obtained also from a tissue with high endogenous RNase activity.