Disulfide bond requirements for active Wnt ligands

Abstract

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.

Keywords: Disulfide; Protein Domain; Wnt Biogenesis; Wnt Pathway; Wnt Signaling; Wnt3a; beta-Catenin.

FIGURE 1.

Sequence alignment of mouse Wnt3a, Xenopus Wnt8, and all human WNT proteins using ClustalW. Identical (black background, white text), conservative (dark gray background, white text), and similar (light gray background, black text) residues are highlighted. Wnt3a exon/intron boundaries are shown above the mature Wnt3a protein sequence. Cysteines 1–24 are numbered based on Wnt3a and highlighted in yellow below the alignment. Green P indicates palmitoleoylation site. The Wnt3a secondary structure shown below the Wnt3a sequence is predicted by PSIPRED. The Xwnt8 secondary structure predicted by PSIPRED and that resolved in the crystal structure (Protein Data Bank entry 4FOA) are shown below the Xwnt8 sequence, respectively: α helices (magenta), β sheets (light blue), and unresolved regions (light gray).

FIGURE 1.

Sequence alignment of mouse Wnt3a, Xenopus Wnt8, and all human WNT proteins using ClustalW. Identical (black background, white text), conservative (dark gray background, white text), and similar (light gray background, black text) residues are highlighted. Wnt3a exon/intron boundaries are shown above the mature Wnt3a protein sequence. Cysteines 1–24 are numbered based on Wnt3a and highlighted in yellow below the alignment. Green P indicates palmitoleoylation site. The Wnt3a secondary structure shown below the Wnt3a sequence is predicted by PSIPRED. The Xwnt8 secondary structure predicted by PSIPRED and that resolved in the crystal structure (Protein Data Bank entry 4FOA) are shown below the Xwnt8 sequence, respectively: α helices (magenta), β sheets (light blue), and unresolved regions (light gray).

FIGURE 2.

Requirement of the individual 24 cysteines of Wnt3a for secretion and/or activity. A, the structure and subdomains of Xwnt8 (Protein Data Bank entry 4FOA) with schematic α helices and β sheets. B, flat diagram showing the 24 cysteines of Wnt3a with the known ensemble of disulfide bonds modeled from the Xwnt8 structure. Note that Xwnt8 lacks the first two conserved cysteines (c1 and c2) found in the very amino-terminal region of most Wnt proteins. C, detection of protein levels of Wnt3a cysteine mutants in the whole cell lysate and in CM using an anti-Wnt3a antibody. Wnt3a in CM was further analyzed using reducing and non-reducing conditions to show the relative amounts of monomeric Wnt3a proteins. Relative density of the Wnt3a protein is quantified in Table 1. D, activity of the Wnt3a cysteine mutants determined by cotransfection with plasmids for Wnt-responsive STF luciferase and control Renilla luciferase reporters. Wnt3a was transfected at 10 ng/well in triplicate. Values were normalized to that of the WT Wnt3a set at 100%. Error bars, adjusted S.D. IB, immunoblot.

FIGURE 3.

Characterization of Wnt3a double cysteine mutants. A, expression of Wnt3a double cysteine mutants in the whole cell lysate and CM using reducing and non-reducing conditions. Asterisks indicate positions of Xwnt8 resolved disulfide bonds. Relative density of the Wnt3a protein is quantified in Table 1. B, activity of the Wnt3a double mutants by cotransfection with the Wnt-responsive STF reporter. C, assessing the dominant negative activity of the Wnt3a c20-c21 mutant through varied dosages of WT and mutant expression constructs as indicated. D, co-immunoprecipitation of the WT Wnt3a or Wnt3a c20-c21 mutants with mFZD8 CRD-Fc. Input CM for the WT Wnt3a and the Wnt3a c20-c21 mutant is shown in the inset. Note that the c20-c21 mutant (red arrow) exhibits a slightly slower mobility than the WT Wnt3a (green arrow). Error bars, S.D. IB, immunoblot; RLU, relative luciferase units.

FIGURE 4.

The conserved Wnt serine residue for palmitoleic acid adduction can be functionally substituted by threonine but not other residues tested. A, Wnt3a Ser-209 mutant expression (50 ng/well) in total cell lysate and in CM. B, the WNT-responsive STF luciferase reporter showing the activity of the WT Wnt3a and the active Wnt3a S209T substitution but the inactivity of S209A, S209G, or S209C substitution. C, similar levels of activity of the WT Wnt3a and Wnt3a S209T at multiple doses. D, Wnt3a mutants harboring substitutions of a portion of the thumb/hairpin 2 from Drosophila WntD (in red). Annotated β sheets above Wnt3a are modeled from the Xwnt8 structure, whereas those above WntD are from the WntD structure. The c9–c12 and c10–c11 disulfide bonds are indicated. E, protein expression levels of Wnt3a/WntD chimera mutants from transfected cells (at 50 ng/well of the expression vector) in total cell lysates and in CM. F, the WNT-responsive STF luciferase reporter showing the activity of the WT Wnt3a and the inactivity of each Wnt3a/WntD chimera mutant, including S209Q. Error bars, S.D. IB, immunoblot; RLU, relative luciferase units.

FIGURE 5.

Generation and characterization of the active V5 tagged WNT proteins. A, comparison of untagged and two versions of V5-tagged WNT3A in non-reducing and reducing conditions. The previous V5(+C linker) and the new V5a constructs code for a WNT3A protein with a predicted addition of 4.4 and 1.9, kDa, respectively. B, the activity of untagged WNT proteins and new WNT-V5a assayed by the TOPFLASH reporter. 50 ng/well of each WNT expression vector was transfected. C, the activity of untagged WNT proteins and the new WNT-V5a in synergy with LRP6 assayed by the TOPFLASH reporter. 50 ng/well of each WNT expression vector plus 1 ng of LRP6 expression vector were co-transfected. D, the activity of untagged WNT proteins and the new WNT-V5a in non-canonical Wnt signaling measured by Dishevelled phosphorylation, which exhibits upward mobility shifts. E, comparison of WNT-V5 (+C linker) and WNT-V5a protein expression in whole cell lysates and CM under reducing conditions. The linker and the V5 tag for the original V5(+C linker) version (+CILKGGRADPAFLYVVDLLGPRFEGKPIPNPLLGLDSTRTG) was replaced by +GGGKPIPNPLLGLDSTRTG in the new WNT-V5a constructs. The V5(+C linker) and V5a add a predicted 4.4 and 1.9 kDa, respectively, to each WNT protein. Note that the WNT2-V5(+C linker) construct used in this experiment contains a frameshift mutation in the linker region that destroys the V5 tag. Error bars, S.D. NSP, nonspecific band in the lysate. IB, immunoblot; RLU, relative luciferase units.