Structure Short Article

Structural Insights into the Inhibition of Wnt Signaling by 5T4/Wnt-Activated Inhibitory Factor 1

Yuguang Zhao,1,3 Tomas Malinauskas,1,2,3 Karl Harlos,1 and E. Yvonne Jones1,* 1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK 2Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA 3These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2014.01.009 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY 5T4) induced an antitumor immune response in mouse cancer models (Woods et al., 2002; Mulryan et al., 2002) and has been The tumor antigen 5T4/WAIF1 (Wnt-activated inhibi- evaluated in clinical trials targeting , renal cell tory factor 1; also known as Trophoblast glycopro- , and refractory prostate cancer (Kim et al., 2010). tein TPBG) is a cell surface protein targeted in multi- For the most comprehensive phase 3 clinical study of MVA- ple cancer clinical trials. Recently, 5T4 carried out to date (in 733 patients with metastatic renal it has been shown that 5T4/WAIF1 inhibits Wnt/ cancer), no difference was observed in survival for the overall b-catenin signaling, a signaling system central to study population, but analyses suggested that subgroups of patients could benefit (Amato et al., 2010). 5T4/WAIF1-targeted many developmental and pathological processes. b -based therapies are also under active development Wnt/ -catenin signaling is controlled by multiple in- (Boghaert et al., 2008; Sapra et al., 2013). hibitors and activators. Here, we report crystal struc- Despite the potential therapeutic value of 5T4/WAIF1 as a tures for the extracellular domain of 5T4/WAIF1 at target in oncology, knowledge of the function and molecular 1.8 A˚ resolution. They reveal a highly glycosylated, mechanism of action of this cell surface molecule remains rigid core, comprising eight leucine-rich repeats sparse. 5T4/WAIF1 is predicted to contain multiple leucine (LRRs), which serves as a platform to present rich-repeats (LRRs) in the extracellular domain (Myers et al., evolutionarily conserved surface residues in the 1994), a transmembrane helix and a cytoplasmic region (Fig- N-terminal LRR1. Structural and cell-based analyses, ure 1A; Figure S1 available online). The cytoplasmic PDZ-binding coupled with previously reported in vivo data, sug- motif Ser-Asp-Val of 5T4/WAIF1 has been reported to interact gest that Tyr325 plus the LRR1 surface centered on with the PDZ domain of TIP-2/GIPC (Awan et al., 2002), a cyto- plasmic protein that associates with vesicles located near the a second exposed aromatic residue, Phe97, are b cell membrane (De Vries et al., 1998). Further downstream essential for inhibition of Wnt/ -catenin signaling. mechanisms of signal transduction remain unknown. Recently, These results provide a structural basis for the devel- 5T4/WAIF1 has been found to inhibit the Wnt/b-catenin signaling opment of 5T4/WAIF1-targeted therapies that pre- pathway (Kagermeier-Schenk et al., 2011), a key pathway in serve or block 5T4/WAIF1-mediated inhibition of embryonic development and a major target for anticancer thera- Wnt/b-catenin signaling. peutics (MacDonald et al., 2009; Polakis, 2012). Kagermeier- Schenk and colleagues (Kagermeier-Schenk et al., 2011) reported that one of three 5T4/WAIF1 paralogs in zebrafish, INTRODUCTION 5T4/Waif1a, co-immunoprecipitated with the Wnt coreceptor LRP6. However, the extracellular domains of 5T4/Waif1a and 5T4/WAIF1 (also known as trophoblast glycoprotein, TPBG; 5T4 LRP6 did not interact directly; this indirect interaction appears oncofetal trophoblast glycoprotein; and Wnt-activated inhibitory to require colocalization of both partners in the membrane factor 1, WAIF1) is a vertebrate-specific, single-pass transmem- and/or bridging by an unknown molecule. brane protein first identified in human placental tissues (Hole and 5T4/WAIF1 is heavily glycosylated (Hole and Stern, 1988) Stern, 1988). 5T4/WAIF1 is rarely expressed in normal adult tis- posing challenges for production in standard Escherichia coli- sues, but is present at high levels in placenta and in most com- based protein overexpression systems. To date, biophysical mon tumors, typically more than 80% of of the studies of 5T4/WAIF1 are lacking. As a first step in elucidating kidney, breast, colon, prostate, and ovary (Hole and Stern, the molecular mechanisms governing 5T4/WAIF1 function and 1988; Southall et al., 1990; Starzynska et al., 1994). Thus, 5T4/ its role in Wnt/b-catenin signaling, we established protocols for WAIF1 has the characteristics of an oncofetal antigen, high- the production of the soluble extracellular domain of 5T4/ lighting it as a possible candidate for use as a diagnostic marker WAIF1 (5T4/WAIF1Ecto) using a human embryonic kidney 293 or target for cancer treatment. A modified vaccinia virus Ankara (HEK293) cell-based stable expression system. We determined

(MVA) encoding human 5T4/WAIF1 (designated TroVax or MVA- three structures of the extracellular domain of 5T4/WAIF1Ecto

612 Structure 22, 612–620, April 8, 2014 ª2014 The Authors Structure Inhibition of Wnt Signaling by Cancer Antigen 5T4

from two crystal forms at 1.75–1.77 A˚ resolution. Our structural, 0.17 A˚ for 218 equivalent Ca atoms from crystal 1 and crystal evolutionary, and cell-based analyses, coupled to previously 2, chain A; 0.25 A˚ (231 atoms) for crystal 1 and crystal 2, chain reported in vivo studies of two zebrafish 5T4/Waif1 paralogs, B(Figure S3). LRRs 7–8 provide a platform onto which the CT- 5T4/Waif1a and 5T4/Waif1c, reveal key residues of 5T4/WAIF1 cap is integrated and contain additional elements (an a helix that are essential for inhibition of the Wnt/b-catenin signaling C300–E311 at the C terminus of LRR 8; Figure 1C). Finally, pathway. both N- and C-terminal caps contain two disulfide bonds each

that further stabilize the seamless LRR core of 5T4/WAIF1Ecto. RESULTS AND DISCUSSION A Potential 5T4/WAIF1-Protein Interaction Hotspot

Production of the Extracellular Domain of 5T4/WAIF1 We investigated the surface characteristics of 5T4/WAIF1Ecto to Human 5T4/WAIF1 contains 420 amino acid residues and is delineate the areas that had properties indicative of interaction a single-pass transmembrane protein (Figures 1A and S1). sites. Specific protein-protein interactions are rarely mediated

Sequence analysis predicts a domain structure comprising by flexible, heterogeneous glycans. 5T4/WAIF1Ecto contains a secretion signal (residues 1–31), serine-rich motif (32–53), seven predicted asparagine-linked glycosylation sites at N81, LRRs (60–344), a transmembrane region (356–376), and a short N124, N166, N192, N243, N256, and N275 (Figures 1A and cytoplasmic tail (377–420; Figure S1). We expressed a full-length S1). Electron density corresponding to five of these seven poten-

LRR region of 5T4/WAIF1 (D60–D345, 5T4/WAIF1Ecto) with a tial glycans is visible in crystal 1 (Figure 1B). The glycan on N166 C-terminal rhodopsin 1D4 tag (Hodges et al., 1988) as a secreted is not visible because it is located in a disordered loop (N166– construct in HEK293 GnTI(À) cells (Reeves et al., 2002; Aricescu P172); however, fragmentary electron density proximal to N166 et al., 2006; Zhao et al., 2011). A stable cell line was generated by is visible in crystal 2, chain B, where loop N166–P172 is stabilized using a PhiC31 integrase-assisted vector with puromycin selec- by a neighboring molecule in the crystal lattice. The glycan on tion, pURD (see Experimental Procedures). We purified 5T4/ N192 is not visible, possibly because of flexibility as the putative

WAIF1Ecto using antibody-affinity chromatography followed by site is exposed in solvent channels for both crystal forms. Thus, size exclusion chromatography. Multi-angle light scattering our crystal structures suggest that at least five of the putative

(MALS) analysis indicated that the purified 5T4/WAIF1Ecto was N-linked glycosylation sites of 5T4/WAIF1Ecto are glycosylated. monomeric in solution (Figure S2A). These five glycosylated asparagines therefore flag positions on the ectodomain structure that are less likely to mediate 5T4/

Crystal Structures of 5T4/WAIF1Ecto Reveal the LRR WAIF1-protein interactions. Core Decorated with Loops and Glycans The concave face of 5T4/WAIF1Ecto contains an extended, Enzymatically deglycosylated 5T4/WAIF1Ecto crystallized in two negatively charged region that spans five LRRs: E177 in LRR4; crystal forms, 1 and 2, with one and two molecules in the asym- E216, LRR5; D240 LRR6; E266 and D267, LRR7; and D292, metric unit, respectively (Table 1). Initial crystallographic phases LRR8 (Figure 1D). This region is bordered by the glycosylated were calculated by molecular replacement using LRRs of Netrin N124 on LRR2 and an array of positively charged residues: G ligand 1 (NGL1; Protein Data Bank [PDB] ID 3ZYJ; Seiradake R213, R214, R237, H238, H264, and R288. These residues et al., 2011) as a search model. Final models from crystal forms bind sulfate ions in crystal form 2 (Figure 1E). Notably, similar

1 and 2 were refined to an Rwork/Rfree of 18.2%/22.2% and positively charged surfaces mediate interactions among Wnts, 17.7%/21.7% at 1.75 A˚ and 1.77 A˚ resolution, respectively. their inhibitors, and heparan sulfate proteoglycans (HSPG; Mali-

All three structures of 5T4/WAIF1Ecto reveal a compact core of nauskas et al., 2011), suggesting that 5T4/WAIF1 interactions eight LRRs. The LRRs build-up a right-handed slightly twisted with its binding partners could be modulated by HSPGs. 5T4/ solenoid in which the short b strands are arranged like a ladder WAIF1Ecto did not bind to a heparin column (Figure S2B), consis- in the concave face of 5T4/WAIF1Ecto (Figures 1B and 1C). The tent with the calculated isoelectric point of 5.36, arguing against eight LRRs are flanked by two, N- and C-terminal, cysteine- 5T4-heparan sulfate interactions. However, this finding does not rich capping modules (NT-cap and CT-cap, respectively) that exclude the possibility that charged regions of 5T4/WAIF1 could complete the overall structural framework of 5T4/WAIF1Ecto. interact with HSPGs and/or other proteins. The eight LRRs could be classified into three distinct groups (Fig- Protein-protein interactions are frequently mediated by rigid, ure 1C). LRRs 1–3 and 6 contain a sequence motif Lxx(x)V/ larger than 600 A˚ 2 surfaces enriched in aromatic, evolutionarily

LxxLxLxxxxLxxL/VxxxxF (where x is any amino acid). In LRRs conserved residues (Moreira et al., 2007). 5T4/WAIF1Ecto has 1–3, the motif provides a scaffold for a rigid hydrophobic core five aromatic side chains (F97, Y224, Y251, F290, and Y325) where three consecutive phenylalanine residues (F111, F138, exposed on its surface (Figure 1F). We further investigated which and F162) rest on the continuous bed of leucines (Figure 1C). surface exposed residues of 5T4/WAIF1 are conserved across LRRs 4 and 5 form a second distinct group because they contain 5T4/WAIF1 family members in vertebrates. Notably, a cluster a buried phenylalanine at different positions compared to LRRs of evolutionarily conserved residues (E67, S69, and K76) is 1–3, and 6. A flexible loop (N166–P172) that links LRR 3 to centered on F97 of LRR1 (Figure 1F). Thus F97 represents a

LRR 4 is ordered only in one out of the three 5T4/WAIF1Ecto potential ‘‘hot spot’’ that might mediate 5T4/WAIF1-protein inter- structures, crystal 2, chain B (Figure S3). Similarly, a flexible actions and/or might be important for the inhibition of Wnt/b-cat- insertion (P185–S194) with a short 310 helix is ordered only in enin signaling by 5T4/WAIF1. Similarly, the aromatic character of crystal 1 (Figures 1B and S3). Other than these two loops, the Y325 is conserved, and mammalian 5T4/WAIF1 sequences and three 5T4/WAIF1Ecto molecules are essentially identical in the zebrafish Waif1a contain either tyrosine or phenylalanine at this two crystal forms with root mean square deviation (rmsd) of position.

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Comparison of 5T4/WAIF1Ecto to Structurally and unique antagonist of Wnt signaling. Our analysis of evolutionarily Functionally Related Proteins conserved surface residues (see previous section) had identified In a second approach to identify where the potential ligand- a putative protein-binding hotspot; therefore, we focused further binding regions of 5T4/WAIF1 are located, we compared 5T4/ analyses onto this region, which encompasses the NT-cap and

WAIF1Ecto to other structures of LRR-containing proteins and LRR1. It contains the surface exposed side chains of S69, E67, their complexes. We used the secondary structure matching T74, K76, N95, and, most notably, F97 (Figures 2A and 2B). program PDBeFold (Krissinel and Henrick, 2004) for protein structure alignment to identify the closest structural homologs Lys76 and Phe97 of 5T4/WAIF1 Are Essential for the of 5T4/WAIF1Ecto. The most similar protein is Netrin G ligand 1 Inhibition of Wnt Signaling (NGL1; PDB ID 3ZYJ, chain A), which binds to Netrin G1 via We used a cell-based Wnt/b-catenin signaling assay (DasGupta its concave surface (Figure 1G; Seiradake et al., 2011). 5T4/ et al., 2005) to investigate the role of the conserved surface res-

WAIF1Ecto and NGL1 could be superimposed based on 222 idues clustered around F97 of human 5T4/WAIF1 (Figure 2). equivalent Ca atoms with an rmsd of 2.0 A˚ , highlighting the over- Based on our crystal structures, we selected residues for site- all conservation of the capped LRR fold. Despite this structural directed mutagenesis that have their side chains exposed on similarity, the sequence identity between 5T4/WAIF1 and the exterior of 5T4/WAIF1 and are therefore less likely to disturb NGL1 is low (10%) and provides no evidence for any similarity the overall structural integrity (Figure 2A). Most full-length, wild- in protein-binding mode. A second structurally similar protein, type and mutant, 5T4/WAIF1 constructs were trafficked to the glycoprotein 1ba (PDB ID 1P9A, chain G; rmsd 2.3 A˚ for 225 cell membrane as assessed by fluorescence microscopy (Fig- equivalent Ca atoms), recognizes its ligand, thrombin, using a ure S4A), except two variants, N124Q and R214E, which were combination of flexible C-terminal tail and two LRRs (Celikel not secreted as 5T4/WAIF1Ecto constructs. Wild-type and et al., 2003). Glycoprotein 1ba wraps around one side of another mutant extracellular domains were expressed and secreted at ligand, von Willebrand factor A1 domain, using N- and C-termi- similar levels (except N124Q and R214E) as assessed by west- nal b-fingers that are absent in 5T4/WAIF1Ecto (Huizinga et al., ern blot and FACS analyses (Figures S4B and S4C), consistent 2002). with them all being folded, glycosylated, and passing cellular

5T4/WAIF1Ecto lacks surfaces or sequence motifs that are pre- quality control systems (Trombetta and Parodi, 2003). sent in other extracellular inhibitors or receptors of the Wnt Consistent with previous studies (Kagermeier-Schenk et al., signaling pathway. It does not have the surface-exposed NxI 2011), conditioned media containing secreted mouse Wnt3a motif that mediates interactions between the Wnt co-receptor activated the Wnt/b-catenin signaling pathway in HEK293T cells, LRP6 and two inhibitors, Dickkopf and Sclerostin (Bourhis and this signaling was inhibited both by the Wnt antagonist Dick- et al., 2011). There is an absence of hydrophobic grooves (pre- kopf (a positive control) and wild-type 5T4/WAIF1 (Figures 2C sent in Wnt8 receptor Frizzled8, Janda et al., 2012) or pockets and S5). Combined mutations of two residues, N338A and (present in the Wnt inhibitory factor 1; Malinauskas, 2008; Mali- A340G, exposed at the CT-cap did not have an effect on the nauskas et al., 2011) that could mediate 5T4/WAIF1Ecto inhibitory inhibitory function of 5T4/WAIF1 (Figures 2A and 2C). Similarly, function by sequestering Wnt-linked palmitoleyl moieties. The combined mutations E189R, R190A, and Q191A in an evolution- only other inhibitor of Wnt signaling that has a LRR fold is Tsu- arily divergent region, the flexible insertion in LRR4 (Figures 1B, kushi, which functions as a Wnt signaling inhibitor by competing 1C, 2A, and 2C), did not affect the 5T4/WAIF1-mediated with Wnt protein for binding to the receptor Frizzled4 (Ohta et al., inhibition. 2011). However, the sequence identity between 5T4/WAIF1 and We investigated two sulfate ion-contacting residues, R214 Tsukushi is relatively low, 20%, suggesting that the mode of and E261, using charge reversal mutations, R214E and E261R, Wnt inhibition may be different. Taken together, our comparison respectively (Figure 1E). E261R did not have an effect on the of 5T4/WAIF1 to other LRR-containing protein recognition mod- Wnt-inhibitory function of 5T4/WAIF1 (Figures 2C and S5), ules and inhibitors of Wnt signaling suggests that 5T4/WAIF1 is a whereas R214E abolished secretion of 5T4/WAIF1Ecto R214E

Figure 1. Crystal Structure of the Human 5T4/WAIF1Ecto

(A) Domain organization of human 5T4/WAIF1. Seven glycosylation sites are marked with hexagons. The 5T4/WAIF1Ecto is colored in blue-to-red transition; serine-rich and transmembrane regions are gray.  (B) Ribbon diagram of 5T4/WAIF1Ecto in two views that differ by a 90 rotation around a vertical axis. 5T4/WAIF1Ecto is colored as in (A). Asn-linked N-acetyl- glucosamines are shown as magenta sticks. Disulfide bonds are shown as gray connected spheres.

(C) The structure-based alignment of LRRs of 5T4/WAIF1Ecto reveals repetitive patterns of leucines (or similar hydrophobic residues, valines, and isoleucine) that build up the framework of 5T4/WAIF1Ecto. LRRs 1–3 and LRR6 form a distinctive group; each of these LRRs contains a buried phenylalanine (pink in the bottom panel), which contributes to the tightly packed hydrophobic core of 5T4/WAIF1Ecto. On top of the LRR-based core, the architecture of 5T4/WAIF1Ecto is further stabilized by hydrogen bonding patterns between multiple, three residue-long b strands (highlighted in gray background).

(D) Electrostatic properties of 5T4/WAIF1Ecto. The protein is shown as solvent-accessible surface colored by electrostatic potential at ± 5 kT/e (red, acidic; blue, basic). The orientation of 5T4/WAIF1Ecto on the left side is the same as in (B, left side). Glycan moieties are shown as yellow sticks. Charged residues discussed in the text are indicated. (E) A highly charged, sulfate-binding region on LRRs 5–8, colored as in (B). Side chains of sulfate-binding residues are labeled and shown as sticks (carbons, gray to orange; nitrogen, blue; sulfur, yellow; oxygen, red). Distances between atoms are shown in angstroms.

(F) The surface of 5T4/WAIF1Ecto is colored by residue conservation (conserved, magenta; variable, cyan). Sequences of 45 members of the 5T4/WAIF1 family were included in the sequence conservation analysis. Five surface-exposed aromatic residues are indicated; only one, F97, is evolutionarily conserved.

(G) Superposition of 5T4/WAIF1Ecto (green) onto Netrin G ligand 1 (NGL1; blue) in complex with Netrin G1 (gray surface) illustrates the potential of the concave face of LRRs to recognize LRR-binding proteins.

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Table 1. X-Ray Crystallography Data Collection and Refinement S4A and 2C, respectively). Consistent with the behavior of Statistics the full-length protein, 5T4/WAIF1Ecto N124Q was not secreted (Figure S4B). 5T4/WAIF1Ecto 5T4/WAIF1Ecto (Crystal Form 1) (Crystal Form 2) Mutation of the highly conserved phenylalanine residue, F97, Data Collection to threonine (F97T) significantly (p = 0.0002) reduced the Wnt- inhibitory function of human 5T4/WAIF1 (Figure 2C). The side X-ray source Diamond Light Diamond Light chain of F97 is sandwiched between the glycosylated N124 Source, beamline I02 Source, beamline I02 and evolutionarily conserved K76 (Figure 2B). The juxtaposition Wavelength (A˚ ) 1.06 1.06 of F97 and K76 could provide further stabilization to this region Resolution range (A˚ ) 49.31–1.75 54.33–1.77 + because of cation-p interactions between the -NH3 group of (1.80–1.75) (1.82–1.77) K76 and the adjacent delocalized electrons of F97, respectively Space group P 21 21 21 P121 1 (Gallivan and Dougherty, 1999). The average distance between Unit cell the nitrogen of the K76 side chain and the carbon atoms of the a, b, c (A˚ ) 49.31, 67.31, 96.38 49.6, 95.82, 65.97 F97 ring is 5.4 A˚ (SD, 0.3 A˚ ) in the two crystal forms. Indeed, a, b, g () 90, 90, 90 90, 91.14, 90 5T4/WAIF1 mutant K76A inhibited Wnt signaling significantly Total reflections 317,855 300,698 (21,206) (p < 0.0001) less efficiently compared to wild-type 5T4/WAIF1 Unique reflections 33,062 59,830 (4,362) in our cell-based assay (Figure 2C). Mutation of the less conserved N95 to alanine did not influence the Wnt-inhibitory Multiplicity 9.6 (9.3) 5.0 (4.9) function. Interestingly, a similar lack of effect on function was Completeness (%) 99.8 (99.2) 99.7 (99.5) observed for a mutation, T74V, of the conserved T74 (Figure 2C). Mean I/sigma (I) 17.9 (3.1) 13.8 (2.5) Thus, while sensitive to mutation of F97 and K76, the inhibitory + À a Rmerge (all I and I ) (%) 6.6 (81.3) 6.3 (76.4) function of 5T4/WAIF1 appears relatively robust to mutation of Refinement neighboring residues (Figures 2B and 2C). Mutants E67A and Resolution range (A˚ ) 43.94–1.75 54.33–1.77 S69A also showed less marked effects on Wnt-inhibitory func- (1.80–1.75) (1.82–1.77) tion than F97T and K76A (Figure 2C). Number of reflections, 31,330/1,674 56,784/3,022 In addition, we tested the effect of mutation Y325A on work/test set the Wnt-inhibitory function of 5T4/WAIF1. Multiple vertebrates Number of atoms 2,493 (2,239/ 4,712 (4,276/ contain phenylalanine at the corresponding position, including (protein/ligands, 130/124) 257/179) zebrafish Wnt-inhibitory Waif1a (F274; zebrafish noninhibitory glycans/waters) Waif1c contains S283 at the equivalent position; Figure S1).

Protein residues 283 552 5T4/WAIF1Ecto Y325A was expressed and secreted at a similar Mean B-factors 36 (35/55/40) 32 (32/52/37) level compared to wild-type 5T4/WAIF1Ecto (Figure S4). The full- (protein/ligands, length 5T4/WAIF1 Y325A did not inhibit Wnt3a signaling in the glycans/waters) (A˚ 2) cell-based assay (Figures 2C and S5), suggesting that, in addi-

Rwork (%) 18.2 (27.9) 17.7 (24.5) tion to the region around F97 and K76, other surfaces of 5T4/ WAIF1 may be important for the inhibition of Wnt signaling. Rfree (%) 22.2 (29.4) 21.7 (29.8) Notably, the surface proximal to Y325 is not obstructed by Rmsd from ideal 0.016 0.016 values (bonds) (A˚ ) Asn-linked glycosylation and thus may be accessible for the in- teractions between 5T4/WAIF1 and currently unknown binding Rmsd from ideal 1.8 1.9 values (angles, ) partners. Our results suggest that residues K76 and F97 are essential Ramachandran plotb for the inhibition of the Wnt/b-catenin signaling pathway by hu- Favored regions (%) 95.7 95.4 man 5T4/WAIF1. The previously reported findings for zebrafish Outliers (%) 0.0 0.2 paralogs 5T4/Waif1a and 5T4/Waif1c (Kagermeier-Schenk Values for the highest resolution shell are shown in parentheses. et al., 2011) shed new light when revisited in the context of a As defined in Aimless (Evans and Murshudov, 2013). our data. Whereas 5T4/Waif1a inhibited Wnt/b-catenin sig- b As defined in MolProbity (Chen et al., 2010). naling, 5T4/Waif1c did not. A chimera comprising the 5T4/ Waif1a ectodomain plus 5T4/Waif1c cytoplasmic domain in- (Figure S4B), suggesting aberrant trafficking of this construct hibited Wnt signaling, whereas the 5T4/Waif1c ectodomain within the cell. Consistent with these observations, 5T4/WAIF1 plus 5T4/Waif1a transmembrane and cytoplasmic regions did R214E did not inhibit Wnt signaling (Figures 2C and S5). We not inhibit. These results indicated that the 5T4/Waif1 cyto- note that positive charge at the position corresponding to plasmic region is dispensable for Wnt signaling inhibition; R214 in human 5T4/WAIF1 is relatively conserved across spe- however, they also raise the question as to which differences cies (Arg/Lys/His in 11 of 15 species; Figure S1), suggesting between the ectodomains of 5T4/Waif1a and 5T4/Waif1c are that the charge characteristics of this position play a role in the responsible for the difference in inhibitory function. We therefore folding and trafficking of 5T4/WAIF1. analyzed the ectodomain sequences (Figure 2D). We found that Similarly to R214E, mutation of glycosylated N124 to the Wnt-inhibitory 5T4/Waif1a in zebrafish has K40, F61, and glutamine impaired trafficking to the cell surface and Wnt- F274 at the positions corresponding to K76, F97, and Y325 in inhibitory function of full-length 5T4/WAIF1 N124Q (Figures human 5T4/WAIF1. In contrast, the noninhibitory 5T4/Waif1c

616 Structure 22, 612–620, April 8, 2014 ª2014 The Authors Structure Inhibition of Wnt Signaling by Cancer Antigen 5T4

Figure 2. An Evolutionarily Conserved Re-

gion of 5T4/WAIF1Ecto Is Essential for the Inhibition of the

(A) The surface of the 5T4/WAIF1Ecto is colored by residue conservation as in Figure 1F. Residues investigated in the Wnt-responsive cell-based assay are indicated. (B) A surface-exposed region that mediates the Wnt-inhibitory function of 5T4/WAIF1. b Strands

of 5T4/WAIF1Ecto are colored as in Figure 1B, numbering corresponds to Figure S1. Nitrogen and oxygen atoms of selected side chains and glycans are shown in blue and red, respectively. Disulfide bonds are shown as gray connected spheres. (C) The inhibition of Wnt3a signaling in a cellular assay by wild-type and mutant constructs of 5T4/ WAIF1 and the Wnt inhibitor Dickkopf. The signaling was induced using conditioned media containing secreted mouse Wnt3a. Wnt3a sig- naling was inhibited by wild-type 5T4/WAIF1 and Dickkopf, but it was significantly less inhibited by 5T4/WAIF1 mutant constructs K76A and F97N. The experiment was repeated three times (results from experiment 1 are shown here, from experi- ments 2 and 3; Figure S5), each time in quadru- plicate, and error bars show SD. The p values were calculated using paired t test are shown for the wild-type K76A, F97T, and Y325A pairs. The other mutant constructs of 5T4/WAIF1 did not show significant inhibition of Wnt signaling as suggested by p values (p > 0.05). Columns corresponding to two mutant constructs of 5T4/WAIF1 that exhibited impaired trafficking to the cell surface (N124Q and R214E; Figure S4) are shown in gray. (D) Amino acid sequence alignment of the N-ter- minal regions of the 5T4/WAIF1 family members. K76 and F97, which are essential for the Wnt- inhibitory function of human 5T4/WAIF1 (Fig- ure 2C), are marked with red stars. Corresponding residues N52 and N73 in the noninhibitory 5T4/ Waif1c from zebrafish are framed in black. Boundaries between the NT-cap, LRR1, and LRR2 are shown below the alignment. An align- ment of the full-length 5T4/WAIF1 proteins is presented in Figure S1. has N52, N73, and S283 at the corresponding positions, further that link 5T4/WAIF1, a tumor antigen that is the focus of multiple supporting the notion that these surface-exposed residues play late phase clinical trials, with the Wnt signaling pathway, a well- a key role in the inhibition of Wnt/b-catenin signaling by 5T4/ established but challenging target in oncology (Polakis, 2012). WAIF1 family members. These data provide a structural basis for the assessment of cur- rent therapeutic anti-5T4/WAIF1 (Boghaert et al., Future Directions and Implications for Cancer Therapies 2008; Sapra et al., 2013) in terms of their ability to preserve or Our structural, evolutionary, and cell-based analyses, combined block 5T4/WAIF1-mediated inhibition of Wnt/b-catenin signaling with previously reported in vivo studies in zebrafish (Kagermeier- with consequent benefits and risks. Schenk et al., 2011), suggest that the conserved surface centered on K76 and F97, plus a second area involving Y325, EXPERIMENTAL PROCEDURES is essential for the inhibition of the Wnt/b-catenin signaling pathway by human 5T4/WAIF1. However, the proteins that Protein Production bind to these surfaces remain to be identified. The protocols We cloned human 5T4/WAIF1 (UniProtKB/Swiss-Prot Q13641) extracellular we report here for the production of wild-type and mutant 5T4/ domain (residues D60–D345) construct 5T4/WAIF1Ecto from the IMAGE clone WAIF1 proteins could provide useful reagents for the identifica- 438906 (Source BioScience) into a stable cell line generation vector pURD that we had recently engineered in-house (Figure S6). HEK293S GnTI(À) cells tion and validation of 5T4/WAIF1 binding partners (cf. the (Reeves et al., 2002) were cotransfected with pURD-5T4/WAIF1Ecto and strategy of So¨ llner and Wright, 2009), to add further intriguing in- a PhiC31 integrase expression vector (pgk-phiC31). The polyclonal cell popu- sights into the varied mechanisms by which Wnt signaling can be lation resulting from puromycin (2 mg/ml) selection was used for the protein modulated. Our studies detail the specific atomic-level features production. The secreted 5T4/WAIF1Ecto protein has additional amino acids

Structure 22, 612–620, April 8, 2014 ª2014 The Authors 617 Structure Inhibition of Wnt Signaling by Cancer Antigen 5T4

from the cloning vector: N-terminal ETG and C-terminal GTETSQVAPA se- Expression Analysis of 5T4/WAIF1 Constructs quences (last nine residues are Rhodopsin 1D4 tag; Molday and MacKenzie, For western blot analyses of secreted 5T4/WAIF1 constructs, residues 32–355 1983). For protein purification, the conditioned media were passed through were cloned into a modified pHLsec vector with a C-terminal 1D4 tag and ex- the anti-1D4 tag antibody (University of British Columbia) covalently linked pressed in HEK293T cells for 2 days. 5T4/WAIF1 samples (cell lysates and to Sepharose beads at 4C (CNBr-activated Sepharose 4 Fast Flow, GE conditioned media) were reduced with dithiothreitol and heated for 5 min  Healthcare) and eluted with 1D4 peptide. For deglycosylation, 5T4/WAIF1Ecto at 100 C before loading on the SDS-PAGE gels. Western blots were probed was treated with endo-b-N-acetylglucosaminidase F1 (37C, 1 hr) and further with mouse anti-1D4 tag (University of British Columbia), anti-His tag (Penta purified by size-exclusion chromatography (Superdex 200 16/60 column, GE His, QIAGEN), anti-a-tubulin (Sigma), and anti-b-actin (Abcam) antibodies, Healthcare) in 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl. followed by goat anti-mouse IgG-horseradish peroxidise (HRP) conjugate (Sigma). HRP was detected using the Amersham ECL western blotting detec- Crystallization and Structure Determination tion kit (GE Healthcare Life Sciences). For the expression analysis of human 5T4/WAIF1 on the cell surface, wild-type and mutant constructs (residues 5T4/WAIF1Ecto was concentrated to 5.5 mg/ml and crystallized using the sitting drop, vapor diffusion method at 21C(Walter et al., 2005). The crystal- 32–420) were cloned into a modified pHLsec vector (pHLsec-mVenus), which has a C-terminal monomeric fluorescent protein mVenus (Nagai et al., 2002) lization drop contained 100 nl of concentrated 5T4/WAIF1Ecto, 100 nl of 25% w/v polyethylene glycol (PEG) 3350, 0.1 M citrate pH 3.5, and 0.5 nl of tag (a gift from A. Clayton and A. R. Aricescu, Oxford) and expressed in 0.1 M NaOH (crystal form 1). For crystal form 2, the crystallization drop con- COS-7 cells for 2 days. Images of the cells were taken using the Leica SP8-X SMD inverted microscope. tained 100 nl of concentrated 5T4/WAIF1Ecto plus 100 nl of 0.2 M (NH4)2SO4, 30% w/v PEG 4000. Crystals were flash-frozen by immersion into a reservoir Flow cytometry analysis of 5T4/WAIF1 tagged with a fluorescent mono- solution supplemented with 25% v/v glycerol followed by transfer to liquid Venus protein was performed in HEK293T cells. The expression of mono- nitrogen. The crystals were kept at À173C during X-ray diffraction data Venus was monitored via flow cytometry on a CyAn ADP Analyzer (Beckman collection. Diffraction data were indexed and integrated using XDS (Kabsch, Coulter). The argon-ion laser was tuned to 488 nm with 100 mW of 2010), and scaled and merged using Aimless (Evans and Murshudov, 2013). power, and mono-Venus fluorescence detected in FL1 through a 530/ Initial X-ray phases were determined by molecular replacement using the 40-nm bandpass filter. Data analysis was performed using FlowJo software NGL1 structure (PDB ID 3ZYJ; Seiradake et al., 2011) as a search model in (Tree Star). Phaser (McCoy et al., 2007). Structures were built using Buccaneer (Cowtan, 2008), PHENIX AutoBuild (Terwilliger et al., 2008), further refined in REFMAC ACCESSION NUMBERS (Murshudov et al., 1997), validated using MolProbity (Chen et al., 2010), and analyzed using ConSurf (Glaser et al., 2003) and the PyMOL Molecular The PDB accession numbers for the structure factors and coordinates corre- Graphics System (Schro¨ dinger). Data collection and refinement statistics sponding to the crystal forms 1 and 2 of 5T4/WAIF1Ecto are 4CNM and 4CNC, are shown in Table 1. respectively.

Multi-Angle Light Scattering Analysis SUPPLEMENTAL INFORMATION MALS analysis of 5T4/WAIF1Ecto was performed during analytical size exclu- sion chromatography using Superdex 75 10/300 GL column (GE Healthcare Supplemental Information includes six figures and can be found with this Life Sciences) connected to static light-scattering (DAWN HELEOS II, Wyatt article online at http://dx.doi.org/10.1016/j.str.2014.01.009. Technology), differential refractive index (Optilab rEX, Wyatt Technology), and Agilent 1200 UV (Agilent Technologies) detectors. Purified 5T4/WAIF1Ecto (2.24 mg/ml, 0.1 ml) was loaded onto a column equilibrated in 150 mM NaCl, ACKNOWLEDGMENTS 10 mM HEPES, at pH 7.5. Data were analyzed using the ASTRA software pack- age (Wyatt Technology). We thank Diamond Light Source for beamtime (proposal mx8423) and the staff of beamline I02 for assistance with crystal testing and data collection. We are Heparin Affinity Chromatography grateful to Thomas Walter for assistance with crystallization, Benjamin Davies for the PhiC31 integrase expression vector, Keith Morris for confocal imaging, Purified 5T4/WAIF1Ecto (0.358 mg/ml, 0.69 ml) was loaded onto a 5 ml HiTrap heparin HP column (GE Healthcare Life Sciences) equilibrated in 40 mM NaCl, Veronica T. Chang for flow cytometry analysis, and members of the Division of Structural Biology for helpful discussion and assistance. This work was funded 10 mM HEPES, and pH 7.5. Elution of 5T4/WAIF1Ecto (flow rate 2 ml/min) was followed by absorption at 280 nm. A linear NaCl gradient, from 40 mM NaCl, by Cancer Research UK, the UK Medical Research Council (A10976 and 10 mM HEPES, pH 7.5 to 1 M NaCl, 10 mM HEPES, pH 7.5 over ten column G9900061 to E.Y.J.) and the Wellcome Trust (grant 090532/Z/09/Z supporting the Wellcome Trust Centre for Human Genetics). Y.Z. was supported by the volumes was applied but 5T4/WAIF1Ecto did not bind to the column and was eluted with 40 mM NaCl, 10 mM HEPES, at pH 7.5. BioStruct-X project funded by the seventh Framework Programme (FP7) of the European Commission. T.M. was supported by the UK Medical Research Council. Cellular Assays for Wnt Signaling Wnt3a-responsive cell-based assays were carried out using HEK293T cells Received: October 15, 2013 in quadruplicate (in 96-well plates, 105 cells/well). Cells were cotransfected Revised: December 30, 2013 with a SuperTopFlash firefly luciferase plasmid (300 ng/well; DasGupta Accepted: January 13, 2014 et al., 2005), a constitutive Renilla luciferase plasmid (pRL-TK from Promega Published: February 27, 2014 100 ng/well), and a 5T4/WAIF1 wild-type (residues 1–420, cloned into the pneo-sec vector via EcoR1 and Xho1 sites; pneo-sec is similar to pHLsec [Aricescu et al., 2006], but it contains a neomycin/kanamycin resistance REFERENCES selection marker) or a mutation variant or Dkk plasmid (Chen et al., 2011; 300 ng/well). Lipofectamine 2000 (Invitrogen) was used according to the Amato, R.J., Hawkins, R.E., Kaufman, H.L., Thompson, J.A., Tomczak, P., manufacturer’s recommendations. Sixteen hours after transfection, the Szczylik, C., McDonald, M., Eastty, S., Shingler, W.H., de Belin, J., et al. media were replaced with the conditioned media from the mouse L-Wnt3a (2010). Vaccination of metastatic renal cancer patients with MVA-5T4: a cell line for Wnt signaling induction. The firefly and Renilla luciferase activities randomized, double-blind, placebo-controlled phase III study. Clin. Cancer were measured 24 hr later with the Dual-Glo luciferase reporter assay Res. 16, 5539–5547. system (Promega) using an Ascent Lunimoskan luminometer (Labsystems). Aricescu, A.R., Lu, W., and Jones, E.Y. (2006). A time- and cost-efficient sys- The enzymatic activity of firefly luciferase was normalized compared to tem for high-level protein production in mammalian cells. Acta Crystallogr. constitutive Renilla luciferase activity. D Biol. Crystallogr. 62, 1243–1250.

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