Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The human transmembrane mucins.
Figure 2: SDS-PAGE and thermal-stability analyses of wild-type and mutant MUC1 SEA samples.
Figure 3: SEA domain sequences and the structure of human MUC1 SEA.
Figure 4: NMR spectroscopy of wild-type SEA and uncleaved SEA mutants.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Hollingsworth, M.A. & Swanson, B.J. Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer 4, 45–60 (2004).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Khatri, I.A., Wang, R.Q. & Forstner, J.F. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Abe, J., Fukuzawa, T. & Hirose, S. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Lillehoj, E.P., Han, F. & Kim, K.C. Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding. Biochem. Biophys. Res. Commun. 307, 743–749 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Google Scholar 

  12. Lopez, X., Mujika, J.I., Blackburn, G.M. & Karplus, M. Alkaline hydrolysis of amide bonds: effect of bond twist and nitrogen pyramidalization. J. Phys. Chem. A 107, 2304–2315 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Linsley, P.S., Kallestad, J.C. & Horn, D. Biosynthesis of high molecular weight breast carcinoma associated mucin glycoproteins. J. Biol. Chem. 17, 8390–8397 (1988).

    Google Scholar 

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

    Google Scholar 

  16. Carter, P. & Wells, J.A. Dissecting the catalytic triad of a serine protease. Nature 332, 564–568 (1988).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Maeda, T. 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).

    Article  CAS  Google Scholar 

  19. Matsui, H. 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).

    Article  CAS  Google Scholar 

  20. Helgstrand, M., Kraulis, P., Allard, P. & Härd, T. Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra. J. Biomol. NMR 18, 329–336 (2000).

    Article  CAS  Google Scholar 

  21. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. & Skelton, N.J. Protein NMR Spectroscopy: Principles and Practice (Academic Press, London, 1996).

    Google Scholar 

  22. Neri, D., Szyperski, T., Otting, G., Senn, H. & Wüthrich, K. 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).

    Article  CAS  Google Scholar 

  23. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  Google Scholar 

  24. Schwieters, C.D., Kuszewski, J.J., Tjandra, N. & Clore, G.M. The Xplor-NIH NMR molecular structures determination package. J. Magn. Reson. 160, 65–73 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Kuszewski, J., Gronenborn, A.M. & Clore, G.M. 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).

    Article  CAS  Google Scholar 

  27. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    Article  CAS  Google Scholar 

  28. Thompson, J.D., Higgins, D.G. & Gibson, T.J. 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).

    Article  CAS  Google Scholar 

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

Authors and Affiliations

Authors

Corresponding author

Correspondence to Torleif Härd.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

SDS-PAGE analyses of wild-type and mutant MUC1 SEA samples. (PDF 416 kb)

Supplementary Fig. 2

Circular dichroism spectra. (PDF 150 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Macao, B., Johansson, D., Hansson, G. et al. Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat Struct Mol Biol 13, 71–76 (2006). https://doi.org/10.1038/nsmb1035

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1035

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing