CHARACTERIZATION OF 2 DIFFERENT GLYCOSYLATED DOMAINS FROM THE INSOLUBLE MUCIN COMPLEX OF RAT SMALL-INTESTINE

The highly glycosylated domains of rat small intestinal mucins were is olated after reduction and trypsin digestion and separated into two po pulations (A and B) by gel chromatography. The molecular mass values w ere 650 and 335 kDa, respectively, and the relative yields suggest tha t the two glycopeptides occur in equimolar proportions. Electron micro scopy revealed linear structures with weight average lengths of 230 nm (A) and 110 nm (B) corresponding to a mass/unit length of about 3 kDa /nm. The protein cores (17-19%) contain large amounts of threonine (ov er 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfa te account for approximately 80 and 0.5%, respectively, and gas chroma tography-mass spectrometry showed that the patterns of neutral and sia lic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc resid ues, the average oligosaccharide is about 4 sugar residues long, and t he largest species observed are heptasaccharides. The major neutral an d sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)Ga1NAcol, respectively. Sialic acid is presen t as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extracti ons of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas e xposure to dithiothreitol or high speed homogenization accomplished co mplete solubilization. The ''subunits'' obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments co rresponding in size to the latter are obtained after cleavage with try psin. Most of the mucins from rat small intestine thus occurs as an in soluble glycoprotein complex composed of subunits joined with disulfid e bonds. The subunits contain two highly glycosylated regions with dif ferent lengths substituted with very similar oligosaccharides.