| Background and Purpose:It was revealed that the colon mucus of mice was composed of two layers with different characteristics. The loose layer on the lumen side could be removed by gentle suction, while the inner layer firmly attached to the epithelia was removed by gentle scraping, indicating the different physical properties between the two layers. Interestingly, FISH (fluorescence in situ hybridization) combined with immunofluorescence assay on the paraffin-embed colon sections of mice displayed the absolutely different distribution patterns of bacteria in the two layers. The loose layer was contaminated with bacteria, whereas the firm layer was free of bacteria, implying the different antimicrobial effects of the two layers.The mechanisms associated with the different properties remain unknown. However, proteomic analyses revealed the identity of proteins in both mucus layers. The Muc2 mucin and other goblet cell-derived molecules such as Fc gamma binding protein(Fcgbp/ IgGFcγBP) and chloride channel calcium activated 3 (clca3) were present in both the loose layer and the firm layer. The MUC2 mucin is the structural constituent of intestinal mucus, and IgGFcγBP is postulated to be involved in immune protection of intestinal epithelia, thus the difference of constituents was not capable of explaining the different characteristics of the two mucus layer convincingly. In the result of proteomics analysis of two mucus layers, the information of TFF3 secreted by goblet cell was missing. As we know, TFFs(trefoil factor family) were believed to be a group of mucin-associated molecules, and different members were found to be co-localized with specific mucins in proper tissues, thus affecting the secretion, stability or viscoelasticity of mucins. In addition, the two molecules were involved in protection of epithelia synergistically. It was showed that rTFF3 and IgGFcγBP were involved in the protection of intestinal epithelia. In DSS-induced colitis of rats, It was revealed, with histoimmunochemistry assay, that the expression level of rIgGFcγBP was similar to that of rTFF3 during the onset, active or regeneration phase respectively. It's known that rTFF3 is an important molecule involved in restitution of epithelia, implying the ability of rIgGFcγBP to protect epithelia and functional association of rIgGFcγBP with rTFF3.Recently, Johansson et al showed that the intact Muc2 was bound to IgGFcγBP with covalent linkage in mice colon mucus. However, it's believed that mucus covering epithelia interacts with trefoil factor family. Here, we plan to find the potential clues associated with the different properties of the two mucus layers via identification of presence, molecular forms of rTFF3, rMuc2 and rIgGFcγBP. The further study is to analyze the interaction of the three molecules above with Co-immunoprecipitation and Western blot assay.Method and material:A mini vacuum pump and glass slides were sequentially used to collect rat colon mucus samples of different layers according to the document. The intact mucosa, post-suction mucosa or post-scraping mucosa was fixed immediately in Carnoy fixtitive at 4℃for 2 hours. Paraffin-embedded sections were stained with AB-PAS so as to validate the reliability of the methods of sample collection. In order to identify the presence of rTFF3 in mucus and its localization pattern compared with rMuc2 and rIgGFcγBP, the antibodies against the proteins above combined with SABC-FITC were used to detect the corresponding antigens distributed in the intact mucus layer. Non-reducing SDS-PAGE or reducing SDS-PAGE followed by Western blot were used to analyze the molecular forms of rTFF3,rMuc2 or rIgGFcγBP present in loose layer, firm layer or single mucosa respectively so as to find the potential clues relevant to different properties of the two mucus layers by identifying the difference of amount or molecular forms of the three proteins. Anti-rTFF3 serum,anti-rMuc2 serum, anti-rIgGFcγBP serum or preimmune serum were used to co-immunoprecipitate the corresponding antigens presensent in rat colon mucus, and each of the co-immunoprecipitated samples was tested by Anti-rTFF3 serum,anti-rMuc2 serum, anti-rIgGFcγBP serum respectively with immunoblot assay to analyze the interaction of rTFF3, rMuc2 and rIgGFcγBP.Resμlt1. AB-PAS staining showed apparent difference of mucus thickness between the intact mucosa and post-suction mucosa, while no mucus was observed after the scraping of post-suction mucosa. The results proved the reliability of the method of sample collection.2. The Immunofluoresence assay showed localization of rTFF3 in the mucus. It was localized in mucus and granules of goblet cell together with rMuc2 and rIgGFcγBP.3.Western blot assay showed the presence of rTFF3 complex(250kD) in both the loose layer and the firm layer, while the monomer(6kD) was present in the loose layer, the firm layer and the single mucosa layer. Besides, the complex seemed to be localized mainly in the loose layer, whereas the trace of the complex was found in the firm layer. Under non-reducing condition, the complex of rMuc2 with a molecular weight of above 250kD was tested in the samples from the loose layer, firm layer and mucosa. Under reducing condition, such a complex shifted to a subunit of 164kD as well as the 278kD subunit in the loose layer and firm layer. rIgGFcγBP were present in form of complex under non-reducing condition, However, the complex shifted to the subunits of 258kD,214kD,140kD respectively when reduced.4. Western blot combined with co-immunoprecipitation showed that anti-rTFF3 serum failed to test the corresponding antigen in the samples co-immunoprecipitated with anti-rMuc2 serum or anti-rIgGFcBP serum, whereas the antigen was tested in the sample co-immunopricipitated with anti-rTFF3 serum itself. anti-rMuc2 serum or anti-rIgGFcBP serum tested the corresponding antigens in both samples co-immunoprecipitated with anti-rMuc2 serum or anti-rIgGFcBP serum, while failed to tested the corresponding antigens in the sample co-immunopricipitated with anti-rTFF3 serum. All anti-serums above failed to test the corresponding antigens in the sample co-immunoprecipitated with the pre-immune serum.ConclusionThe goblet cell-derived proteins rTFF3, rMuc2 or rIgGFcγBP were all localized in the mucus layer of rat colon. rTFF3 was present in the loose mucus layer in form of complex, which may be associated with the different characteristics of different mucus layers; rMuc2, rIgGFcγBP were present in both mucus layers in form of complex and shifted to subunits when reduced. rIgGFcγBP, but not rTFF3 was shown to interact with rMuc2, which was in line with the previous result based on proteomics analyses.
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