UBC Theses and Dissertations
Degradation of rat colonic epithelial glycoproteins by cell free extracts of rat faeces Poon, Hiron Hay-Lun
The luminal surface of the gastrointestinal tract is coated with a thick, mucilaginous epithelial glycoprotein containing mucus whose functions are believed to include both lubrication of the faecal stream and protection of the underlying mucosal epithelial cells. Chemical and histochemical investigations have shown that the colonic epithelial glycoproteins of man and rat contain terminal non-reducing sialic acid residues, the great majority of which have 0-acetyl substituents located at position C₄ and/or on the polyhydroxy side chain. Although the biological role of such sialic acids is unknown the observations that (a) 4-O-acetyl sialic acids are resistant to bacterial neuraminidases; (b) sialic acids protect some glycoproteins against proteolysis; and (c) most faecal, bacterial, enzymes are exo-qlycosidases have led to the suggestion that O-acetylated sialic acids may contribute to the "integrity of the mucus barrier" by retarding the degradation of the mucus glycoproteins. In the present study, the degradation of the purified colonic epithelial glycoproteins of Wistar rat colon, bovine submandibular mucin, human seromucoid and rat seromucoid, and the hydrolysis of artificial p-nitrophenyl substrates by the enzymes present in fresh faeces from Wistar rats is reported. Sterile, cell free, extracts of Wistar rat faeces in a "minimal media" at pH7.0 contain (i) exo-glycosidases capable of hydrolyzing the α-linked L-fucose and the β-linked D-galactose, N-acetyl-D-glucosamine and probably the N-acetyl-D-galactosamine residues of rat colonic epithelial glycoproteins; (ii) neuraminidase(s) capable of removing sialic acids both with or without side chain substituents from bovine submandibular mucin and rat colonic epithelial glycoproteins; and (iii) an esterase which removes O-acetyl substituents from the side chain of sialic acid residues. Studies of the removal of sialic acids from bovine submandibular mucin and rat colonic epithelial glycoproteins indicated that: (i) the rate of removal of O-acetylated sialic acids was apparently dependent upon the 4-O-acetylated sialic acid content of the substrate; (ii) sialic acids were removed more rapidly from de-O-acetylated glycoproteins; and (iii) the faecal enzymes remove a greater proportion of the sialic acid of both the de-O-acetylated and native glycoproteins than was removed with Vibrio cholera neuraminidase. This suggests that the removal of sialic acids is mediated by the esterase induced de-O-acetylation of 4-O-acetyl sialic acids, together with the exo-glycosidase catalyzed cleavage of carbohydrate residues which sterically inhibit neuraminidase activity. However, the possibility of a neuraminidase capable of cleaving 4-O-acetyl sialic acid residues has not been eliminated. Enzymes were also detected which (i) hydrolyzed p-nitrophenyl glycosides and p-nitrophenyl acetate; and (ii) destroyed N-acetyl neuraminic acid. The results of the study demonstrated that the degradation of colonic epithelial glycoproteins by the faecal enzyme extracts from Wistar rat apparently involved the combined actions of glycosidases, neuraminidase, and an esterase. This suggested that changes in either the proportion and/or the activity of the various faecal enzymes could lead to the breakdown of the otherwise normal mucus barrier. Therefore, small changes in the bacterial flora in disease such as ulcerative colitis could be of considerable significance.
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