Disulfide bond requirements for active Wnt ligands.
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.
['MacDonald|Bryan T|BT|', 'Hien|Annie|A|', 'Zhang|Xinjun|X|', 'Iranloye|Oladoyin|O|', 'Virshup|David M|DM|', 'Waterman|Marian L|ML|', 'He|Xi|X|']
D000595:Amino Acid Sequence D000818:Animals D018360:Crystallography, X-Ray D003545:Cysteine D004220:Disulfides D006801:Humans D008024:Ligands D051379:Mice D008969:Molecular Sequence Data D011485:Protein Binding D016415:Sequence Alignment D015398:Signal Transduction D060509:Wnt3A Protein
Disulfide Wnt3a Wnt Signaling Protein Domain Wnt Biogenesis Wnt Pathway beta-Catenin