The isolation of a high-molecular-weight glycoprotein from pig colonic mucus.

Abstract

The principal components of a variety of mucous secretions have been found to be glycoproteins, many of mol.wt. 500000 or more (Gottschalk, 1972). We have isolated the glycoprotein from pig colonic mucus and examined its structure in order to compare it with that of the well-characterized glycoprotein of the gastric mucus from the same species (Snary & Allen, 1971 ; Allen, 1977). Mucosal scrapings were prepared from the washed colons of freshly slaughtered pigs. After exhaustive dialysis of the scrapings against water containing 0.02 % azide, the water-soluble mucus was separated from the insoluble mucous gel and cell debris by squeezing through muslin followed by centrifugation (Fig. 1). The water-soluble mucus, which represented 70% dry wt. of the non-diffusible mucus, contained 72% by wt. protein, 10% by wt. nucleic acid and 4.6% by wt. glycoprotein. Nucleic acid was completely removed by digestion of the water-soluble mucus (concentration 4-7 mg/ml) with proteinase-free bovine pancreatic ribonuclease (EC 3.1.4.22; final concentration 15 pg/ml) and ox pancreatic deoxyribonuclease (EC 3.1.4.5; final concentration 2.5,ug/ml). Incubation was for 17 hat 37C, after which all the nucleic acid was diffusible. The non-covalently bound protein was separated from the glycoprotein by equilibrium density-gradient centrifugation in CsCl (Starkey et al., 1974). Owing to the high ratio of non-covalently bound protein to glycoprotein, it was necessary to repeat this step (on the recovered glycoprotein) in order to achieve complete separation of the two (Fig. 1). The purified glycoprotein was subsequently fractionated into a high-molecularweight glycoprotein A (excluded) and a low-molecular-weight glycoprotein fraction B (included) by gel filtration on Sepharose 4B. The high-molecular-weight glycoprotein A was the principal component, accounting for 84 % of the glycoprotein recovered from the Sepharose column. The low-molecular-weight minor component, the glycoprotein fraction B, has a similar ratio of sugars to glycoprotein A (Table 1) but a higher protein content (40 % by wt.). The high-molecular-weight glycoprotein A had a sugar analysis characteristic of glycoproteins from other mucous secretions (Gottschalk, 1972) with both sulphate and sialic acid present (Table 1). The glycoprotein possessed A blood-group activity (1.25,ug/ 0.lml; assayed by the method of Kabat & Bezer, 1945) which was doubled (0.63,ug/ 0.1 ml) by removal of the sialic acid residues with neuraminidase. A full complement of amino acids was present, although threonine, serine and proline comprised 54 % of the total residues. It is noteworthy that the blood-group activity and both the sugar and the amino acid composition of the colonic glycoprotein are similar to that of the glycoprotein from pig gastric mucus (Starkey et al., 1974), except that the colonic glycoprotein has more sialic acid and less fucose and galactose. Further similarities between the glycoproteins from the two mucous secretions were apparent after Pronase digestion. The Pronase-digested glycoprotein from colonic mucus was separated from the peptides and added Pronase by equilibrium density-gradient centrifugation in CsCl. Analysis of the Pronase-digested glycoprotein showed that carbohydrate had been conserved, but 29.8% of the peptide was lost. Threonine, serine and proline comprised 75% of the remaining amino acids, showing a proportionately bigger loss in the other amino acids. This results points to the presence of non-glycosylated and glycosylated regions of the peptide core in the colonic glycoprotein similar to that found for glycoproteins from other mucous secretions (Donald, 1973 ; Scawen &Allen, 1975). The presence of sialic acid and sulphate together in the same glycoprotein has been shown for other glycoproteins from mucous secretions, for example sheep colonic mucus (Kent & Marsden, 1963) and pig gastric mucus (Starkey et al., 1974).