Article | Published:

Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin

Nature Structural & Molecular Biology volume 13, pages 7176 (2006) | Download Citation

Subjects

Abstract

The single cell layer of the lungs and the gastrointestinal tract is protected by the mucus formed by large glycoproteins called mucins. Transmembrane mucins typically contain 110-residue SEA domains located next to the membrane. These domains undergo post-translational cleavage between glycine and serine in a characteristic GSVVV sequence, but the two peptides remain tightly associated. We show that the SEA domain of the human MUC1 transmembrane mucin undergoes a novel type of autoproteolysis, which is catalyzed by conformational stress and the conserved serine hydroxyl. We propose that self-cleaving SEA domains have evolved to dissociate as a result of mechanical rather than chemical stress at the apical cell membrane and that this protects epithelial cells from rupture. We further suggest that the cell can register mechanical shear at the mucosal surface if the dissociation is signaled via loss of a SEA-binding protein.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Protein Data Bank

References

  1. 1.

    & Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer 4, 45–60 (2004).

  2. 2.

    et al. Cell-associated episialin is a complex containing two proteins derived from a common precursor. J. Biol. Chem. 267, 6171–6177 (1992).

  3. 3.

    et al. Identification of MUC1 proteolytic cleavage sites in vivo. Biochem. Biophys. Res. Commun. 283, 715–720 (2001).

  4. 4.

    , & SEA (Sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3. Biochem. J. 372, 263–270 (2003).

  5. 5.

    et al. The role of the SEA module in cleavage of membrane-tethered mucins. FEBS J. 272, 2901–2911 (2005).

  6. 6.

    , & Cleavage of Ig-Hepta at a “SEA” module and at a conserved G protein-coupled receptor proteolytic site. J. Biol. Chem. 277, 23391–23398 (2002).

  7. 7.

    et al. Sialomucin complex, a heterodimeric glycoprotein complex. J. Biol. Chem. 271, 33476–33485 (1996).

  8. 8.

    et al. The MUC1 SEA module is a self-cleaving domain. J. Biol. Chem. 280, 33374–33386 (2005).

  9. 9.

    , & Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding. Biochem. Biophys. Res. Commun. 307, 743–749 (2003).

  10. 10.

    et al. Structural constraints on protein self-processing in L-aspartate-α-decarboxylase. EMBO J. 22, 6193–6204 (2003).

  11. 11.

    in Structure and Mechanism in Protein Science Ch. 18 and 19, 540–614 (W.H. Freeman and Co., New York, USA, 1999).

  12. 12.

    , , & Alkaline hydrolysis of amide bonds: effect of bond twist and nitrogen pyramidalization. J. Phys. Chem. A 107, 2304–2315 (2003).

  13. 13.

    & Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 118, 6105–6109 (1996).

  14. 14.

    , & Biosynthesis of high molecular weight breast carcinoma associated mucin glycoproteins. J. Biol. Chem. 17, 8390–8397 (1988).

  15. 15.

    & Biosynthesis of MAM-6, an epithelial sialomucin. J. Biol. Chem. 9, 4215–4222 (1988).

  16. 16.

    & Dissecting the catalytic triad of a serine protease. Nature 332, 564–568 (1988).

  17. 17.

    et al. ProTherm, thermodynamic database for proteins and mutants: developments in version 3.0. Nucleic Acids Res. 30, 301–302 (2002).

  18. 18.

    et al. Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16). J. Biol. Chem. 279, 13174–13182 (2004).

  19. 19.

    et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95, 1005–1015 (1998).

  20. 20.

    , , & Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra. J. Biomol. NMR 18, 329–336 (2000).

  21. 21.

    , , & Protein NMR Spectroscopy: Principles and Practice (Academic Press, London, 1996).

  22. 22.

    , , , & Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989).

  23. 23.

    , & Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

  24. 24.

    , , & The Xplor-NIH NMR molecular structures determination package. J. Magn. Reson. 160, 65–73 (2003).

  25. 25.

    , & Improving the packing and accuracy of NMR structure with a pseudopotential for the radius of gyration. J. Am. Chem. Soc. 121, 2337–2338 (1999).

  26. 26.

    , & Improvements and extensions in the conformational database potential for the refinement of NMR and X-ray structures of proteins and nucleic acids. J. Magn. Reson. 125, 171–177 (1997).

  27. 27.

    , & MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

  28. 28.

    , & CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

Download references

Acknowledgements

This work was supported in part by grants from the Swedish Research Council, the Hasselblad Foundation and the Wallenberg Foundation (to the Swedish NMR Centre).

Author information

Affiliations

  1. Department of Medical Biochemistry, Göteborg University, Box 440, SE-405 30 Göteborg, Sweden.

    • Bertil Macao
    • , Denny G A Johansson
    • , Gunnar C Hansson
    •  & Torleif Härd
  2. The Swedish NMR Centre, Göteborg University, Box 440, SE-405 30 Göteborg, Sweden.

    • Torleif Härd

Authors

  1. Search for Bertil Macao in:

  2. Search for Denny G A Johansson in:

  3. Search for Gunnar C Hansson in:

  4. Search for Torleif Härd in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Torleif Härd.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    SDS-PAGE analyses of wild-type and mutant MUC1 SEA samples.

  2. 2.

    Supplementary Fig. 2

    Circular dichroism spectra.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb1035

Further reading