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Disulfide bond requirements for active Wnt ligands.

Publication ,  Journal Article
MacDonald, BT; Hien, A; Zhang, X; Iranloye, O; Virshup, DM; Waterman, ML; He, X
Published in: J Biol Chem
June 27, 2014

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

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Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

June 27, 2014

Volume

289

Issue

26

Start / End Page

18122 / 18136

Location

United States

Related Subject Headings

  • Wnt3A Protein
  • Signal Transduction
  • Sequence Alignment
  • Protein Binding
  • Molecular Sequence Data
  • Mice
  • Ligands
  • Humans
  • Disulfides
  • Cysteine
 

Citation

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MacDonald, B. T., Hien, A., Zhang, X., Iranloye, O., Virshup, D. M., Waterman, M. L., & He, X. (2014). Disulfide bond requirements for active Wnt ligands. J Biol Chem, 289(26), 18122–18136. https://doi.org/10.1074/jbc.M114.575027
MacDonald, Bryan T., Annie Hien, Xinjun Zhang, Oladoyin Iranloye, David M. Virshup, Marian L. Waterman, and Xi He. “Disulfide bond requirements for active Wnt ligands.J Biol Chem 289, no. 26 (June 27, 2014): 18122–36. https://doi.org/10.1074/jbc.M114.575027.
MacDonald BT, Hien A, Zhang X, Iranloye O, Virshup DM, Waterman ML, et al. Disulfide bond requirements for active Wnt ligands. J Biol Chem. 2014 Jun 27;289(26):18122–36.
MacDonald, Bryan T., et al. “Disulfide bond requirements for active Wnt ligands.J Biol Chem, vol. 289, no. 26, June 2014, pp. 18122–36. Pubmed, doi:10.1074/jbc.M114.575027.
MacDonald BT, Hien A, Zhang X, Iranloye O, Virshup DM, Waterman ML, He X. Disulfide bond requirements for active Wnt ligands. J Biol Chem. 2014 Jun 27;289(26):18122–18136.

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

June 27, 2014

Volume

289

Issue

26

Start / End Page

18122 / 18136

Location

United States

Related Subject Headings

  • Wnt3A Protein
  • Signal Transduction
  • Sequence Alignment
  • Protein Binding
  • Molecular Sequence Data
  • Mice
  • Ligands
  • Humans
  • Disulfides
  • Cysteine