Search

A glycoprotein was isolated from rat-colonic mucosa. Analytical ultracentrifugation studies showed the glycoprotein to be homogeneous, having an apparent molecular weight of 9.0 X 10(5); no subunits could be detected in the presence of sodium dodecyl sulfate. It contained 14% of protein and 86% of carbohydrate. The principal sugars in the glycoprotein were galactose, fucose, sialic acid, 2-acetamido-2-deoxygalactose, and 2-acetamido-2-deoxyglucose. A small proportion of mannose was also present. The glycoprotein, apart from the usual carbohydrate constituents present in mucus glycoproteins, contained sulfate, but no uronic acid. High amounts of serine and threonine, and low contents of aromatic and traces of sulfur-containing amino acids, reflect a similarity of this glycoprotein to other mammalian mucus glycoproteins; it differs, however, by its high proportions of Asx + Glx (26 mol.%). Cleavage studies with alkaline borohydride indicated O-glycosidic linkages between N-acetylhexosamine and serine, and threonine, of the peptide core in the glycoprotein. Only about one third of the serine and threonine was linked to the carbohydrate side-chains, which averaged about 22 units in length and were apparently branched.

The carbohydrate side chains of mucus-type glycoproteins are O-glycosidic bonds between N-acetylgalactosamine to the hydroxyl groups of serine and threonine in the protein core. The alkaline catalyzed beta-elimination reaction, in the presence of sodium borohydride, is used for determining the number of side chains. The present paper presents a study of the quantitativeness of the alkaline borohydride procedure, using four parameters: the loss of seryl and threonyl residues, the formation of alanine and 2-aminobutanoic acid; the decrease in N-acetylhexosamine and the recovery of the amino sugar alcohols. Bovine, ovine and porcine submandibular glycoproteins were studied. Evidence is presented for the existence of N-acetylglucosamine involvement in O-glycosidic linkages to serine and threonine. Results for the relative rates of beta-elimination indicate that serine-linked glycosides are released more rapidly than threonine-linked glycosides.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.

A purified glycoprotein, designated RSL-major, was isolated from the rat sublingual gland by means of the procedure of Tettamanti and Pigman. It was found to be homogeneous by analytical ultracentrifugation, to have a mol. wt. of 2-2 times 10-6, and to contain 81 percent (W/W) of carbohydrate, which consists mainly of sialic acid, 2-acetamido-2-deocy-D-glucose, 2-acetamido-2-deocy-D-galactose, and D-galactose in the molar ratio of 1.4:1.4:1.0:1.5; small amounts of fucose and mannose [1.2 and 2.8 percent (W/W), respectively] were also present. The sialic acid residues were resistant to the action of V. cholerae neuraminidase. This resistance was completely abolished by removal of the O-acetyl groups contained in the sialic acid. The sialic acid in RSL-major appeared to be a mixture of N-acetyl-4-O-acetyl- and N-acetyl-4, 7(8)-di-O-acetylneuraminic acids. The carbohydrate to protein attachment of RSL-major was shown, by alkaline beta-elimination reaction, to consist of an O-glycosyl linkage between 2-acetamido-2-deoxy-D-galactosyl residues in the oligosaccharide chains and seryl and threonyl residues in the protein core. The average oligosaccharide, contained in RSL-major, was postulated to be a heptasaccharide. A second material, designated RSL-minor, and also isolated from the ratsublingual gland, was obtained as a mixture of glycoprotein(s) and hydroxylapatite gel, and was not purified further.