Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica

Tse, Sil-King; Chadee, Kris

doi:10.1128/iai.60.4.1603-1612.1992

Cited by 24 publications

(7 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Electrophoresis of mucins, staining and fluorography. Sodium dodecyl sulfate polyacrylamide gel elctrophoresis (SDS-PAGE) of labeled and unlabelled mucins was performed under reducing conditions as previously described [42]. Briefly.…”

Section: Preparation Of Crude Secreted Mucinsmentioning

confidence: 99%

“…Characterization of secretory mucins from human colon and colonic adenocarcinoma cells. Human colonic secretory mucins from surgical specimens and from the culture supernatants of LS174T, T84 and Caco-2 cells were fractionated by S4B gel chromatography to isolate the high molecular weight glycoproteins [42]. As shown in Fig.…”

Section: Muc2 and Muc3 Mrna Expression By Colonic Adenocarcinoma Cellmentioning

confidence: 99%

See 1 more Smart Citation

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Göttke

Keller

Belley

et al. 1998

J Eukaryotic Microbiology

View full text Add to dashboard Cite

Mucins secreted from the gastrointestinal epithelium from the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. 3H-glucosamine and 3H-threonine metabolically labeled proteins separated as high M, mucins in the void (Vo > 10(6) Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.

show abstract

Section: Preparation Of Crude Secreted Mucinsmentioning

confidence: 99%

Section: Muc2 and Muc3 Mrna Expression By Colonic Adenocarcinoma Cellmentioning

confidence: 99%

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Göttke

Keller

Belley

et al. 1998

J Eukaryotic Microbiology

View full text Add to dashboard Cite

show abstract

“…The molecular mechanism of mammalian target cell destruction by the E. histolytica trophozoites is not well defined. This process is proposed to include secretagogue-mediated depletion of the enteric mucus (45) and adhesion of trophozoites to the surface of the enterocytes (28,33), followed by target cell lysis (14). These cytopathic effects have been ascribed to a direct contact between trophozoites and mammalian cells (32), although specific proteolytic enzymes may also participate in this process (37).…”

mentioning

confidence: 99%

Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture

et al. 1995

View full text Add to dashboard Cite

In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4؇C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAcspecific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAcspecific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells. MATERIALS AND METHODSTrophozoites. Trophozoites of E. histolytica HM1:IMSS (provided by H. Vermeersch, Laboratory of Pharmaceutical Technology, University of Ghent, Ghent, Belgium) were grown axenically at 35.5ЊC in 15-ml screw-cap tubes with TYI-S-33 medium (6), supplemented with 500 g of piperacillin per ml and 125 g of amikacin per ml. Trophozoites were harvested at the end of the logarithmic growth phase (72 h) by chilling at 4ЊC. They were concentrated at 200 ϫ g for 5 min and used immediately. For some experiments, freshly harvested trophozoites were fixed for 20 min in 3% paraformaldehyde (PF), washed in phosphatebuffered saline (PBS; pH 7.4) containing 0.9 mM Ca 2ϩ and 0.334 mM Mg 2ϩ (PBS Dϩ ), and quenched for 10 min with NH 4 Cl (50 mM in PBS E [Eisen formulation]). Thereafter, they were handled in the same way as live trophozoites. Conditioned medium was prepared from (i) logarithmic-growth-phase cultures in TYI-S-33; (ii) TYI-S-33, with or without serum, that had been incubated for 1 h with the total number of cells from an equal volume of a logarithmic-phase culture; and (iii) cocultures of trophozoites with mammalian cells as described below. Trophozoite cell extracts were prepared by one of the following methods: (i) intermittent sonication (Vibra Cell VC50; Sonics & Materials, Danbury, Conn.) of 2 ϫ 10 6 live trophozoites in 0.5 ml of PBS Dϩ on ice for a total of 45 s or (ii) vortexing...

show abstract

“…The use of closed intestinal loops for injecting E. histolytica trophozoites is a well-established model to study amebic pathogenesis. This model has been used to determine the role of E. histolytica in mucin exocytosis (59) and antimicrobial peptide production (23) in the colon and for biochemical characterization of rat mucins (60). Here closed ileal loops inoculated with E. histolytica were used to quantify Paneth cell localization (lysozyme expression) and function (Crp secretion).…”

Section: Methodsmentioning

confidence: 99%

Entamoeba histolytica Alters Ileal Paneth Cell Functions in Intact and Muc2 Mucin Deficiency

et al. 2018

View full text Add to dashboard Cite

Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth cells contribute to innate host defense in the ileum. Antimicrobial factors including lysozymes and β-defensins are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet cells that form the protective mucus layer critical in gut homeostasis and pathogen invasion. In this study we investigated ileal innate immunity against ), the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in and littermates and quantifying Paneth cell localization (lysozyme expression) and function (Crps secretion). Relative to littermates, showed disorganized mislocalization of Paneth cells that was diffusely distributed with elevated lysozyme secretion in the crypts and on villi in response to Inhibiting Gal-lectin binding with exogenous galactose andCP5- had no effect on parasite-induced erratic Paneth cell lysozyme synthesis. Although basal ileal expression of genes was unaffected in mice in response to there was robust release of pro-inflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, secreted cysteine proteinases cleaved the pro-region of Crp 4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates localization and function of Paneth cells critical in host defense against microbes.

show abstract

Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica

Cited by 24 publications

References 37 publications

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture

Entamoeba histolytica Alters Ileal Paneth Cell Functions in Intact and Muc2 Mucin Deficiency

Contact Info

Product

Resources

About