Susan A. Wagner

The roles of enteric bacterial sialidase, sialate O-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or moreO-acetyl esters at positions C7–C9 on the sialic acids retarded the rate of hydrolysis. A specific sialateO-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and aKM of approximately 1mm sialateO-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and aKM of approximately 1mm.Faecal extracts from ulcerative colitis (UC) patients had higher sialateO-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged.Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acidO-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×105 Da and aggregates in excess of 106 Da, while the minor mucin ranged from 3.0 × 105 to 106 Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.