Abstract
The highly glycosylated domains of rat small intestinal mucins were isolated after reduction and trypsin digestion and separated into two populations (A and B) by gel chromatography. The molecular mass values were 650 and 335 kDa, respectively, and the relative yields suggest that the two glycopeptides occur in equimolar proportions. Electron microscopy 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 (over 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfate account for approximately 80 and 0.5%, respectively, and gas chromatography-mass spectrometry showed that the patterns of neutral and sialic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc residues, the average oligosaccharide is about 4 sugar residues long, and the largest species observed are heptasaccharides. The major neutral and sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)GalNAcol, respectively. Sialic acid is present as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extractions of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas exposure to dithiothreitol or high speed homogenization accomplished complete solubilization. The "subunits" obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments corresponding in size to the latter are obtained after cleavage with trypsin. Most of the mucins from rat small intestine thus occurs as an insoluble glycoprotein complex composed of subunits joined with disulfide bonds. The subunits contain two highly glycosylated regions with different lengths substituted with very similar oligosaccharides.
Highlights
Mucins are long, linear and flexible moleculeswith a central of about 3 kDa/nm
The precipitates were recovered by centrifugationin a bench-top centrifuge, visualized in the electron microscope (Fig. 2), both glycopeptides appeared as linear and apparently flexible structures
Repeated extractions of the intestinal mucosa with guani- jected to gel chromatography on Sephacryl 5-500 eluted dinium chloride left an insoluble residue (RI-Gu) which ac- mainly with the void volumeand after trypsin digestion, the counted for approximately 80% of the total mu- major glycosylatedfragments eluted at the same positions as cins, measured as high molecular mass mucin glycopeptides glycopeptidesA and B on Sephacryl S-500 (Fig. 9)
Summary
-.- were collected and analyzed for hexose (- -) and sialic acid (-). The bars indlcate fractions pooled. After high speed an aliquot (200pl) was taken from the middle of the centrifuge tube centrifugation (18,000 rpm; 30 min; Beckman JA-20 rotor), the su- into a light scattering cell Another aliquot (700 p l ) was diluted and pernatant was dialyzed extensively against water and freeze-dried. The material (-625mg)was dissolved in 4.0 M guanidinium until solutions containing glycopeptideA at 4.36, 2.18, 1.09, and 0.44 chloride (50 ml) and precipitated with ethanol (4 volumes,95%).The mg/ml and glycopeptide B at 4.32, 2.16, 1.08, and 0.43 mg/ml were precipitate was collected by centrifugation in a bench-top centrifuge, obtained. Extrapolations according to Zimm to zero angle dialyzed against water, and freeze-dried This material (195 mg) was and concentration were performed to calculate the values of molecular dissolved in reduction buffer and thewhole procedure of reduction/ weight. Fractions and B-Lyophilized glycopeptides(20 mg) were dissolvedin 10 mlof
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