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

Abstract

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 of N-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 of N-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 of N-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 x 10(5) Da and aggregates in excess of 10(6) Da, while the minor mucin ranged from 3.0 x 10(5) to 10(6) 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.

O‐glycan biosynthesis in human colorectal adenoma cells during progression to cancer

“…The culture medium and cell pellets from the three cell lines were collected and mixed with 4 M guanidine hydrochloride, 5 mM dithiothreitol in NaCl/P, (154 mM NaC1, 10 mM sodium phosphate) pH 7.0 and a mixture of protease inhibitors as described before [31]. Mucin was purified from these cells by density gradient centrifugation in CsCl taking material of density 1.35 -1.45 g/ml fractionated by Sepharose CL 2B chromatography in 4 M guanidine hydrochloride, NaCl/P, [31].…”

“…Mucin was purified from these cells by density gradient centrifugation in CsCl taking material of density 1.35 -1.45 g/ml fractionated by Sepharose CL 2B chromatography in 4 M guanidine hydrochloride, NaCl/P, [31]. The material excluded on the gel filtration step was taken for further analysis.…”

“…The excluded fractions were pooled and further characterized. SDSPAGE on 3% gels was as described [31] with 16-cm or 5-cm gels in BioRad Protean I1 or mini-Protean I1 systems. Samples for autoradiography contained at least 15000 cpm 35S or 50000 cpm 3H/lane.…”

“…Western blots were made from gels with a sheet of Whatman no.1 filter paper between the gel and the Immobilon membrane to prevent the piperazine diacrylamide gel from sticking to the membrane. The transfer was controlled with rainbow markers and was made in a BioRad Transblot apparatus for 14 h at 30 V and 2-3 h at 70 V. Components were detected on Western blots by periodic acid/Schiff stain [31] and with peroxidase-conjugated wheat germ agglutinin.Slot blots were carried out using a Bio-Rad slot blot manifold. Each purified much was applied as 5 pg and 20 pg (dry mass) on slot blots and was positive when probed with peroxidase-conjugated wheat germ agglutinin.…”

Secreted and Transmembrane Mucins Inhibit Gene Transfer with AAV4 More Efficiently than AAV5

“…Bovine submandibular gland mucin (Sigma) contains a major glycoprotein (82% glycosylated) and a minor glycoprotein (65% glycosylated). Both proteins are glycosylated with O-linked carbohydrate only, and nearly all sites contain sialic acid (37). Mucin was adsorbed onto 96-well enzyme-linked immunosorbent assay plates (Corning Costar) for 1 h at 37°C in 5-fold dilutions starting from 1 mg/ml.…”