Glycoconjugate Journal

, Volume 13, Issue 5, pp 823–831

Molecular characterization of the large heavily glycosylated domain glycopeptide from the rat small intestinal Muc2 mucin

  • Niclas G. Karlsson
  • Malin Ev Johansson
  • Noomi Asker
  • Hasse Karlsson
  • Sandra J. Gendler
  • Ingemar Carlstedt
  • Gunnar C. Hansson
Papers

Abstract

The largest high-glycosylated domain, glycopeptide A, of the ‘insoluble’ mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedtet al., 1993,J Biol Chem268: 18771–81). A rabbit antiserum raised against deglycosylated glycopeptide A was used to clone part of a mucin showing homology to the human MUC2 mucin (Hanssonet al., 1994,Biochem Biophys Res Commun198: 181–90). This serum specifically stained goblet cells (paranuclear) in the mouse small intestine. The size of the coding sequence of glycopeptide A was estimated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal parts of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the primers were subtracted. This is slightly larger than the size estimated earlier by biochemical studies. The mRNA encoding the rat Muc2 was slightly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the ‘insoluble’ mucin of the rat small intestine is encoded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A were either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 ofN-acetylglucosamine linked to C-6 of theN-acetylgalactosaminitol as revealed by tandem mass spectrometry of the perdeuteroacetylated oligosaccharides. Eighteen oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowledge of the core peptide of the rat intestinal goblet cell mucin and provide a relatively complete picture of the glycosylation of a defined mucin domain.

Keywords

mucin rat small intestine sulfated oligosaccharides mmunolocalization goblet cell Muc2 

Abbreviations

gpA

glycopeptide A

α-gpA

antiserum against glycopeptide A

CID

collision induced dissociation

FAB

fast atom bombardment

Hex

hexose

HexNAc

N-acetylhexosamine

HexNAcol

N-acetylhexosaminitol

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References

  1. 1.
    Carlstedt I, Karlsson H, Sundler F, Fransson L-Å (1982)Adv Exp Med Biol 144: 155–57.Google Scholar
  2. 2.
    Carlstedt I, Herman A, Karlsson H, Sheehan J, Fransson L-Å, Hansson GC (1993)J Biol Chem 268: 18771–81.Google Scholar
  3. 3.
    Hansson GC, Baeckström D, Carlstedt I, Klinga-Levan K (1994)Biochem Biophys Res Commun 198: 181–90.Google Scholar
  4. 4.
    Gum JR, Hicks JW, Sack TL, Kim YS (1990)Cancer Res 50: 1085–91.Google Scholar
  5. 5.
    Gum JR, Hicks JW, Toribara NW, Rothe EM, Lagace RE, Kim YS (1992)J Biol Chem 267: 21375–83.Google Scholar
  6. 6.
    Gum JR, Hicks JW, Toribara NW, Siddiki B, Kim YS (1994)J Biol Chem 269: 2440–46.Google Scholar
  7. 7.
    Xu G, Huan L, Khatri IA, Wang D, Bennick A, Fahim REF, Forstner GG, Forstner JF (1992)J Biol Chem 267: 5401–7.Google Scholar
  8. 8.
    Xu G, Huan L, Khatri I, Sajjan US, McCool D, Wang D, Jones C, Forstner G, Forstner J (1992)Biochem Biophys Res Commun 183: 821–28.Google Scholar
  9. 9.
    Ohmori H, Dohrman AF, Gallup M, Tsuda T, Kai H, Gum JR, Kim YS, Basbaum CB (1994)J Biol Chem 269: 17833–40.Google Scholar
  10. 10.
    Gendler SJ, Spicer AP (1995)Ann Rev Physiol 57: 607–34.Google Scholar
  11. 11.
    Baeckström D, Hansson GC (submitted)Glycoconjugate J. Google Scholar
  12. 12.
    Sambrook J, Fritsch EF, Maniatis T (1989)Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  13. 13.
    Karlsson NG, Karlsson H, Hansson GC (1995)Glycoconjugate J 12: 69–76.Google Scholar
  14. 14.
    Asker N, Baeckström D, Axelsson MAB, Carlstedt I, Hansson GC (1995)Biochem J 308: 873–80.Google Scholar
  15. 15.
    Domon B, Costello CE (1988)Glycoconjugate J 5: 387–409.Google Scholar
  16. 16.
    Chang SK, Dohrman AF, Basbaum CB, Ho SB, Tsuda T, Toribara NW, Gum JR, Kim YS (1994)Gastroenterology 107: 28–36.Google Scholar
  17. 17.
    Mårtensson S, Lundblad A, Hansson GC, Carlstedt I (1988)Scand J Clinical Invest 48: 633–40.Google Scholar
  18. 18.
    Carlstedt I, Herrmann A, Lindell G, Nordman H (1995)Glycoconjugate J 12: 495.Google Scholar
  19. 19.
    Mawhinney TP, Landrum DC, Gayer DA, Barbero GJ (1992)Carbohydr Res 235: 179–97.Google Scholar
  20. 20.
    Lo-Guidice JM, Wieruszeski JM, Lemoine J, Verbert A, Roussel P, Lamblin G (1994)J Biol Chem 269: 18794–813.Google Scholar
  21. 21.
    Strecker G, Wieruszeski J-M, Martel C, Montreuil J (1987)Glycoconjugate J 4: 329–37.Google Scholar
  22. 22.
    CarbBank (1995) University of Georgia, WA.Google Scholar

Copyright information

© Chapman & Hall 1996

Authors and Affiliations

  • Niclas G. Karlsson
    • 1
  • Malin Ev Johansson
    • 1
  • Noomi Asker
    • 1
  • Hasse Karlsson
    • 1
  • Sandra J. Gendler
    • 2
  • Ingemar Carlstedt
    • 3
  • Gunnar C. Hansson
    • 1
  1. 1.Department of Medical BiochemistryGöteborg UniversityGothenburgSweden
  2. 2.Mayo Clinic ScottsdaleScottsdaleUSA
  3. 3.Department of Medical and Physiological ChemistryLundSweden

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