Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 May 1;243(3):631–640. doi: 10.1042/bj2430631

Characterization and localization of the putative 'link' component in rat small-intestinal mucin.

R E Fahim 1, R D Specian 1, G G Forstner 1, J F Forstner 1
PMCID: PMC1147906  PMID: 3311021

Abstract

Rat intestinal mucin is polymerized by a putative 'link' component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.

Full text

PDF
631

Images in this article

634Fig. 1.
on p.634
634Fig. 2.
on p.634
636Fig. 3.
on p.636
637Fig. 4.
on p.637
638Fig. 5.
on p.638
638Fig. 6.
on p.638
639Fig. 7.
on p.639

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen A. Structure of gastrointestinal mucus glycoproteins and the viscous and gel-forming properties of mucus. Br Med Bull. 1978 Jan;34(1):28–33. [PubMed] [Google Scholar]
  2. Bhaskar K. R., Reid L. Application of density gradient methods for the study of mucus glycoprotein and other macromolecular components of the sol and gel phases of asthmatic sputa. J Biol Chem. 1981 Jul 25;256(14):7583–7589. [PubMed] [Google Scholar]
  3. Carlstedt I., Lindgren H., Sheehan J. K. The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion. Biochem J. 1983 Aug 1;213(2):427–435. doi: 10.1042/bj2130427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Creeth J. M., Bhaskar K. R., Horton J. R., Das I., Lopez-Vidriero M. T., Reid L. The separation and characterization of bronchial glycoproteins by density-gradient methods. Biochem J. 1977 Dec 1;167(3):557–569. doi: 10.1042/bj1670557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dunstone J. R., Morgan W. T. Further observations on the glycoproteins in human ovarian cyst fluids. Biochim Biophys Acta. 1965 Nov 1;101(3):300–314. doi: 10.1016/0926-6534(65)90009-6. [DOI] [PubMed] [Google Scholar]
  6. Fahim R. E., Forstner G. G., Forstner J. F. Heterogeneity of rat goblet-cell mucin before and after reduction. Biochem J. 1983 Jan 1;209(1):117–124. doi: 10.1042/bj2090117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forstner J. F., Ofosu F., Forstner G. G. Radioimmunoassay of intestinal goblet cell mucin. Anal Biochem. 1977 Dec;83(2):657–665. doi: 10.1016/0003-2697(77)90070-7. [DOI] [PubMed] [Google Scholar]
  8. Hanisch F. G., Uhlenbruck G. Structural studies on O- and N-glycosidically linked carbohydrate chains on Collocalia mucin. Hoppe Seylers Z Physiol Chem. 1984 Feb;365(2):119–128. doi: 10.1515/bchm2.1984.365.1.119. [DOI] [PubMed] [Google Scholar]
  9. Henderson L. E., Oroszlan S., Konigsberg W. A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction. Anal Biochem. 1979 Feb;93(1):153–157. [PubMed] [Google Scholar]
  10. Kopp W. L., Trier J. S., Mackenzie I. L., Donaldson R. M., Jr Antibodies to intestinal microvillous membranes. I. Characterization and morphologic localization. J Exp Med. 1968 Aug 1;128(2):357–373. doi: 10.1084/jem.128.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Levine M. J., Herzberg M. C., Ellison S. A., Shomers J. P., Sadowski G. A. Biochemical and immunological comparison of monkey (Macaca arctoides) and human salivary secretions. Comp Biochem Physiol B. 1978;60(4):423–431. doi: 10.1016/0305-0491(78)90072-x. [DOI] [PubMed] [Google Scholar]
  13. Mantle M., Allen A. A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain [proceedings]. Biochem Soc Trans. 1978;6(3):607–609. doi: 10.1042/bst0060607. [DOI] [PubMed] [Google Scholar]
  14. Mantle M., Forstner G. G., Forstner J. F. Antigenic and structural features of goblet-cell mucin of human small intestine. Biochem J. 1984 Jan 1;217(1):159–167. doi: 10.1042/bj2170159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mantle M., Forstner G. G., Forstner J. F. Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis. Biochem J. 1984 Dec 1;224(2):345–354. doi: 10.1042/bj2240345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mantle M., Mantle D., Allen A. Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges. Biochem J. 1981 Apr 1;195(1):277–285. doi: 10.1042/bj1950277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  18. Pearson J. P., Allen A. A protein of 70 000 molecular weight is joined by disulphide bridges to pig gastric-mucus glycoprotein [proceedings]. Biochem Soc Trans. 1980 Jun;8(3):388–389. doi: 10.1042/bst0080388. [DOI] [PubMed] [Google Scholar]
  19. Pearson J. P., Allen A., Parry S. A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure. Biochem J. 1981 Jul 1;197(1):155–162. doi: 10.1042/bj1970155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Prakobphol A., Levine M. J., Tabak L. A., Reddy M. S. Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva. Carbohydr Res. 1982 Oct 1;108(1):111–122. doi: 10.1016/s0008-6215(00)81896-0. [DOI] [PubMed] [Google Scholar]
  21. Roukema P. A., Oderkerk C. H., Salkinoga-Salonen M. S. The murine sublingual and submandibular mucins. Their isolation and characterization. Biochim Biophys Acta. 1976 Apr 23;428(2):432–440. doi: 10.1016/0304-4165(76)90051-9. [DOI] [PubMed] [Google Scholar]
  22. Slomiany A., Slomiany B. L., Witas H., Aono M., Newman L. J. Isolation of fatty acids covalently bound to the gastric mucus glycoprotein of normal and cystic fibrosis patients. Biochem Biophys Res Commun. 1983 May 31;113(1):286–293. doi: 10.1016/0006-291x(83)90464-3. [DOI] [PubMed] [Google Scholar]
  23. Snary D., Allen A., Pain R. H. Structural studies on gastric mucoproteins: lowering of molecular weight after reduction with 2-mercaptoethanol. Biochem Biophys Res Commun. 1970 Aug 24;40(4):844–851. doi: 10.1016/0006-291x(70)90980-0. [DOI] [PubMed] [Google Scholar]
  24. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  25. Tabachnik N. F., Blackburn P., Cerami A. Biochemical and rheological characterization of sputum mucins from a patient with cystic fibrosis. J Biol Chem. 1981 Jul 25;256(14):7161–7165. [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Woods K. R., Wang K. T. Separation of dansyl-amino acids by polyamide layer chromatography. Biochim Biophys Acta. 1967 Feb 21;133(2):369–370. doi: 10.1016/0005-2795(67)90078-5. [DOI] [PubMed] [Google Scholar]
  28. Zanetta J. P., Breckenridge W. C., Vincendon G. Analysis of monosaccharides by gas-liquid chromatography of the O-methyl glycosides as trifluoroacetate derivatives. Application to glycoproteins and glycolipids. J Chromatogr. 1972 Jul 5;69(2):291–304. doi: 10.1016/s0021-9673(00)92897-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES