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.
From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
Bryan T
BT
MacDonald
From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
Annie
A
Hien
From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
Xinjun
X
Zhang
From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
Oladoyin
O
Iranloye
the Program in Cancer and Stem Cell Biology, Duke-National University of Singapore Graduate Medical School, 8 College Road, 169857 Singapore, and.
David M
DM
Virshup
the Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697.
Marian L
ML
Waterman
From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, xi.he@childrens.harvard.edu.
Xi
X
He
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D015398:Signal Transduction; D060509:Wnt3A Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D008024:Ligands; C555510:WNT3A protein, human; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D003545:Cysteine
Disulfide; Protein Domain; Wnt Biogenesis; Wnt Pathway; Wnt Signaling; Wnt3a; beta-Catenin