Structure of the Protein Core of the Glypican Dally-Like and Localization of a Region Important for Hedgehog Signaling

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Min-Sung Kima, Adam M. Saundersb, Brent Y. Hamaokaa,1, Philip A. Beachyb,2, and Daniel J. Leahya,2

aDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bDepartment of Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, and Howard Hughes Medical Institute, Stanford University Schoolof Medicine, Stanford, CA 94305

Contributed by Philip A. Beachy, June 23, 2011 (sent for review April 25, 2011).

Glypicans are heparan sulfate proteoglycans that modulate the to these factors as well as to establish their proper distribution signaling of multiple growth factors active during animal develop- (9, 10, 12, 16–21). The heparan sulfate attachments of glypicans ment, and loss of glypican function is associated with widespread are clearly important for mediating interactions with these growth developmental abnormalities. Glypicans consist of a conserved, factors and downstream signaling components (22, 23), but approximately 45-kDa N-terminal protein core region followed recent work has demonstrated a role for the N-terminal protein by a stalk region that is tethered to the cell membrane by a glyco- domain, which lacks heparan sulfate modifications, in mediating syl-phosphatidylinositol anchor. The stalk regions are predicted to responsiveness to at least Wnt and Hh signals (23–26). be random coil but contain a variable number of attachment sites Curiously, glypicans appear able to play both positive and for heparan sulfate chains. Both the N-terminal protein core and negative roles in mediating Hh signaling. The protein region the heparan sulfate attachments are important for glypican func- of Dally-like contributes positively to Drosophila Hh responsive- tion. We report here the 2.4-Å crystal structure of the N-terminal ness, and the developmental defects in omodysplasia, particularly protein core region of the Drosophila glypican Dally-like (Dlp). This the bone growth defects, are suggestive of a positive role for structure reveals an elongated, α-helical fold for glypican core glypican-6 function in response to Indian hedgehog (7). Notably, regions that does not appear homologous to any known structure. glypican-4 and glypican-6 are most similar to Dlp (vs. Dally) and The Dlp core protein is required for normal responsiveness to complement Dlp function in a Drosophila cultured cell-based Hh Hedgehog (Hh) signals, and we identify a localized region on signaling assay (25). In contrast, the protein region of glypican-3, the Dlp surface important for mediating its function in Hh signal- which is more similar to Dally than Dally-like, is a negative reg- ing. Purified Dlp protein core does not, however, interact appreci- ulator of Hh responsiveness in the mouse (24, 25, 27, 28). Based ably with either Hh or an Hh:Ihog complex. on sequence homology and functional phenotypes, it has thus been speculated that the two major subfamilies of glypicans have lypicans are heparan sulfate proteoglycans (HSPGs) that evolved opposing activities in Hh signal responsiveness (25). Gconsist of an approximately 450 amino acid N-terminal pro- To investigate the molecular basis for glypican function, we tein domain followed by an approximately 100 amino acid stalk have undertaken structural and functional characterization of region that is attached to the outer cell membrane via a glycosyl- the N-terminal protein domain of Dlp and report here its 2.4-Å phosphatidylinositol anchor (1). The N-terminal domain of most crystal structure. We show that the N-terminal protein domains of glypicans is proteolytically processed by a furin-like convertase glypicans adopt an elongated α-helical structure with no evident to produce two chains that remain connected by disulfide bonds homology to any known structure. We have used structure-guided (2). This processing appears required for some but not all glypi- mutagenesis to identify a localized region on the Dlp surface im- can activity (2, 3). The stalk regions of glypicans are predicted portant for the ability of Dlp to mediate Hh signal response. – to be largely random coil and typically contain 1 5 heparan sul- These results are most consistent with Dlp functioning as a bind- fate attachment sites (1, 4). Six glypicans are present in humans ing protein in Hh signaling, but we are unable to detect high- and mice (glypican-1, -2, -3, -4, -5, and -6); two are present in affinity interactions between Dlp and either Hh or an Hh:Ihog Drosophila [Dally and Dally-like (Dlp)] (1). Based on sequence complex. These results establish a molecular basis for mapping similarity, glypicans assort into two subfamilies with glypican-1, and comparing functional regions of different glypicans. -2, -4, -6, and Dlp in one family and glypican-3, -5, and Dally in another (1). Results Glypicans are active in development in both vertebrates A fragment of the Drosophila melanogaster Dally-like protein that and invertebrates. Loss of Dally in fruit flies results in defects encompasses its N-terminal globular region and is fully functional in brain, eye, wings, antennae, and genitalia (5). Loss of in assays of Hh responsiveness (DlpΔNCF) (25) was expressed in glypican-3 in humans is responsible for Simpson–Golabi–Behmel overgrowth syndrome, in which widespread visceral and skeletal abnormalities are present along with a predisposition to tumor Author contributions: M.-S.K., A.M.S., B.Y.H., P.A.B., and D.J.L. designed research; M.-S.K., A.M.S., and B.Y.H. performed research; M.-S.K., A.M.S., B.Y.H., P.A.B., and D.J.L. formation (6). Loss of glypican-6 has recently been shown to analyzed data; and M.-S.K., P.A.B., and D.J.L. wrote the paper. cause omodysplasia, a genetic disorder characterized by variable The authors declare no conflict of interest. heart defects, cognitive delay, skeletal and facial abnormalities, Freely available online through the PNAS open access option. and shortness of stature (7). Much of the function of glypicans Data deposition: The atomic coordinates and structure factors have been deposited in is attributable to modulation of signaling by several heparin- the Protein Data Bank, www.pdb.org (PDB ID code 3ODN). binding growth factors active during development including 1Present address: Department of Chemistry and Biochemistry, University of California, members of the fibroblast growth factor, Hedgehog (Hh), San Diego, La Jolla, CA 92093. β – Wnt, and transforming growth factor- families (8 15). Each 2To whom correspondence may be addressed. E-mail: [email protected] or pbeachy@ of these factors functions as a morphogen to elicit distinct stanford.edu. concentration-dependent responses within target cells, and glypi- This article contains supporting information online at www.pnas.org/lookup/suppl/ cans have been shown to be required both for normal response doi:10.1073/pnas.1109877108/-/DCSupplemental.

13112–13117 ∣ PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1109877108 Downloaded by guest on September 30, 2021 Table 1. Data collection and refinement statistics Native SeMet Space group C2 C2 Cell dimensions, Å a ¼ 97.02, b ¼ 66.42, a ¼ 96.75, b ¼ 66.29, c ¼ 85.73, β ¼ 104.85° c ¼ 84.38, β ¼ 103.76° Peak Remote Wavelength, Å 0.97929 0.97929 0.96406 Resolution, Å 30–2.4 30–2.8 30–2.8 Rsym* 9.9 (63.6) 5.7 (26.2) 5.5 (26.1) Unique reflections 20,563 12,923 12,861 Mean I∕σðIÞ 13.42 (2.19) 18.5 (2.58) 19.61 (2.4) Completeness, % 99.1 (99.9) 89.8 (55.2) 92.2 (62.5) Refinement R ∕R † 24 69∕29 82 work free ,% . . Number of atoms Protein 2,934 Water 69 RMSD Bond lengths, Å 0.01 Bond angles, ° 1.15 Ramachandran Most favored 313 (94.6%) Allowed 16 (4.8%) Generously allowed 2 (0.6%) Disallowed 0 The values in parentheses are for highest-resolution shell. ¼ ∑ jIð Þ − hIð Þij∕∑ ð Þ *Rsym hkl hkl hkl hkl hkl . † ¼ ∑ j − j∕∑ ¼ 5% Rcrys hkl Fobs Fcalc hklFobs; Rfree test set .

dhfr−∕− CHO cells (29), purified, and crystallized. The structure are apparent. A region of positive electrostatic surface potential of DlpΔNCF was determined by multiwavelength anomalous dif- is present on the M lobe (Fig. S2), but DlpΔNCF binds only weakly fraction using crystals of selenomethionyl-substituted DlpΔNCF. to heparin agarose, from which it elutes in approximately The native DlpΔNCF structure was refined with diffraction data 300 mM NaCl. extending to 2.4 Å (Table 1). α DlpΔNCF adopts a cylindrical, all -helical structure approximately 110 Å in length and 30 Å in diameter for which automated homology searches find no structural homologs (Fig. 1A) (30). A B C309 Although three stretches of polypeptide traverse the long axis N N C317:C553 S-S bond of DlpΔNCF, concerted kinks or breaks in long helices and asso- ciations with shorter helices define three lobes within the α C306 DlpΔNCF structure. We term these lobes the N lobe (N-terminal α segment of α1, α6, α7, and α13), M lobe (middle segment of α1; N-lobe α C-terminal segment of α5, α8, α12, and α14), and C lobe (C-terminal segment of α1, α2, α3, α4; N-terminal segment of α5, α9, α10, α C296:C328 C321:C538 α α BIOPHYSICS AND and 11) based on the region of 1 contained in the lobe (Fig. 1A). S-S bond S-S bond

Electron density for much of the N lobe is poor, and residues COMPUTATIONAL BIOLOGY 74–119, 570–571, and 577–588 in this region are not modeled, R α α which may contribute to a higher than desirable free (Table 1). Nonetheless, the high fraction of alpha helix, the identification of M-lobe α 14∕17 methionine positions from anomalous scattering, and the quality of electron density maps in modeled regions provide con- C C fidence in the model. Six of the seven disulfide bonds conserved in glypicans map to the N lobe and one to the C lobe, but one of α the N lobe disulfides and partners of two N-lobe cysteines are not α α visible in electron density maps and unmodeled (Fig. 1B). The C-terminal chain of DlpΔNCF that results from furin-like proces- C-lobe α sing does not form an independent structural unit but instead contributes helices to the N and M lobes (Fig. 1B). Two disulfide α C241:C392 bonds connect the N- and C-terminal segments, consistent S-S bond α with SDS-PAGE analysis of reduced and nonreduced DlpΔNCF α (Fig. 1B). The C terminus of DlpΔNCF does not emerge from an end of the molecule but rather from the boundary between the M and C lobes (Fig. 1). The level of amino acid sequence conservation among N-terminal glypican core regions suggests Fig. 1. DlpΔNCF structure. (A) Ribbon diagram of the DlpΔNCF structure all glypicans share these structural features (Fig. S1). colored with a rainbow gradient from the N (blue) to the C terminus (red). The termini are labeled, and the N, M, and C lobes are indicated. The DlpΔNCF structure provides no evidence for a functional A dashed line connects the termini generated by furin-like processing of role for Dlp beyond serving as a binding partner. No active DlpΔNCF.(B) Ribbon diagram of DlpΔNCF in which the N-terminal fragment site-like cavities are present, and no sources of conformational is colored slate blue and the C-terminal fragment red. The positions of flexion that could reflect or transmit different activity states cysteines and disulfide bonds are indicated.

Kim et al. PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32 ∣ 13113 Downloaded by guest on September 30, 2021 A B We showed previously that DlpΔNCF fails to interact with HhN Group13 Group12 in a pull-down assay with purified proteins (25). All structural evidence suggests Dlp functions as a binding protein, however, Group14 and a form of Dlp lacking heparan attachments was reported to coimmunoprecipitate with HhN from cultured cells (26). These results suggest that an additional factor or factors present Group9 Group1 in the cell lysates used in coimmunoprecipitation experiments

180° may be needed to promote high-affinity interactions between Group8 DlpΔNCF and HhN. The adhesion-like molecule Ihog was recently shown to bind HhN in a heparin-dependent manner and function Group2 – Group6 Group3 as an essential coreceptor for HhN (31 33). We thus tested Group10 whether Ihog is able to promote interactions between DlpΔNCF and HhN. Addition of an active fragment of Ihog encompassing Group4 Group7 Group5 its two type III fibronectin repeats, IhogFn12, failed to promote Group11 interactions between DlpΔNCF and HhN, however, although stoichiometric amounts of IhogFn12 bound to HhN (Fig. 4A). Furthermore, we were unable to demonstrate an interaction Fig. 2. Clusters of Dlp surface mutations. (A) The positions of the 14 clusters between ShhN and the purified protein domains of either mam- of Dlp surface mutations tested for their effects on the ability of Dlp to med- malian glypican-3 or glypican-6 in a pull-down assay (Fig. 4B). iate Hh responsiveness are indicated. The orientation of DlpΔNCF in the left panel is the same as the ribbon diagram in Fig. 1A. The orientation in the left Discussion panel was rotated 180° about a vertical axis to generate the orientation in the Glypicans modulate the activity of multiple growth factors active right panel. (B) The positions of the 10 DlpΔNCF residues that when collectively mutated eliminate Dlp function in Hh responsiveness are shown colored red. during development, and defects in glypican function lead to widespread and diverse developmental malformations (1, 5–7, 34). Much of the activity of glypicans can be attributed to inter- To identify functionally important regions of Dlp, the DlpΔNCF structure was used to design fourteen clusters of alanine actions between their heparan sulfate attachments and heparan- mutations that collectively blanket the DlpΔNCF surface (Fig. 2A binding growth factors, but recent work has demonstrated impor- and Table S1). Each of these fourteen Dlp variants was tant functional roles for the protein cores of glypicans, notably in constructed and assayed for its ability to restore Hh signaling in Hh and Wnt signaling (24–26, 35). In particular, the protein cores cells in which expression of endogenous Dlp is knocked down. All seem likely to mediate functions that are specific to particular fourteen Dlp variants expressed well (Fig. S3), and four showed glypicans. We report here the crystal structure of the N-terminal diminished ability to mediate Hh signaling (Fig. 3A). The four globular region of a glypican, DlpΔNCF, and show it to adopt an mutation clusters that affect Dlp function map to adjacent elongated, all α-helical fold with no evident homology to pre- regions on the C lobe, but none results in complete loss of activity. viously determined structures. The high level of sequence conser- Subsets of adjacent residues from these clusters (G4p, G5p, and vation among the N-terminal protein cores of glypicans—greater G6p), indicated in bold in Table S1, were thus combined into new than 40% sequence identity exists between Drosophila and — clusters, and a 10-residue grouping of mutations on α4 and α5in human glypicans in this region indicates that the DlpΔNCF struc- the C lobe (alanine substitutions for D235, E236, N237, R240, ture provides a sound basis for the design and interpretation of E244, H245, E248, K250, D254, and K258) was found that results experiments with other glypicans (Fig. S1). The absence of any in a complete loss of the ability of Dlp to mediate Hh apparent active site-like cavities or sources of conformational signaling (Figs. 2B and 3B). This cluster contains several residues flexibility in the DlpΔNCF structure suggests that the protein that are conserved in human glypican-4 and glypican-6, which regions of glypicans exert their effects by serving as binding complement Dlp in Hh signaling, but not human glypican-3, proteins, consistent with proposed roles as coreceptors or in tar- which does not (Fig. S1) (25). geting ligands to specific subcellular compartments (25, 26, 28).

A 12 B 14 Hh+ Hh+ Hh- Hh- 10 12

10 8

8 6 6 4 4 2

Normalized ptc-luciferase activity 2 Normalized ptc-luciferase activity

0 0 Expression gfp gfp gfp gfp dlp G1 G2 G3 G4 G5 G6 G7 G8G9 G10 G11 G12 G13 G14 Expression gfp gfp gfp gfp dlp G6p G4p G4p+G5p construct: ∆GAG construct: ∆GAG +G5p +G6p dsRNA: yfp ptc smo dlp UTR dsRNA: yfp ptc smo dlp UTR

Fig. 3. Dlp function in Hh responsiveness. (A)Aptc-luciferase Hh reporter assay shows the relative abilities of the fourteen Dlp surface variants to mediate Hh responsiveness. dsRNA was cotransfected to knockdown endogenous Dlp or control proteins (gfp, green fluorescent protein; yfp, yellow fluorescent protein; ptc, Patched; smo, Smoothened; ∆GAG, heparan sulfate attachment sites mutated). The G4, G5, G6, and G7 mutants have less than 70% activity of Dlp∆GAG. The residues changed in each mutant group are listed in Table S1.(B) A combined subset of Group4 (G4p) and Group5 (G5p) mutations are sufficient to block the Hh response almost completely. Residues mutated in the G4p, G5p, and G6p clusters are shown in bold in Table S1.

13114 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1109877108 Kim et al. Downloaded by guest on September 30, 2021 Immobilized concentration (36), and a multivalent ligand greatly increases the A HhN resin Blank resin avidity of binding (37–39). Interactions between a monovalent HhN + --+++--- ligand and cell-surface components may thus not be strong IhogFN12 - + - + - ++- + enough to be observed with soluble components in solution. Dlp --+ - ++- ++ Our inability to observe an interaction between ShhN and the Deca-Heparin +CaCl2 ---++++++ N-terminal domain of glypican-3 is puzzling, however, given that KDa the protein region of glypican-3 has been reported to bind to ShhN with nanomolar affinity (24). Several possibilities may 100 75 explain this discrepancy: (i) ShhN may interact with the glypican-3 stalk region, which was present in the earlier study but 50 not in our experiment; (ii) attachment of histidine-tagged ShhN 37 to Ni-NTA agarose may have blocked a glypican interaction site in our studies; or (iii) an unidentified cofactor that promotes 25 high-affinity interaction was present in the earlier studies but ab- 20 sent in our studies. Calcium ions, for example, are required to 15 promote high-affinity interactions between ShhN and CDO (40). 10 Our ability to identify a localized region on the C lobe of the Dlp surface important for proper Dlp function in Hh signaling is B Immobilized Shh resin further consistent with Dlp participating in Hh signaling primarily CDO FNIII + --+ --+ -- as a binding protein, although the nature and number of binding Mouse Gpc6 - + --+ --+ - partners remains to be determined. Curiously, the identified sur- Human Gpc3 + + + face is composed largely of hydrophilic residues, which is unusual ------– CaCl2 ---+++--- for protein protein interfaces (41). This surface also occurs on EGTA ------+++ the opposite surface of Dlp relative to the disordered Dlp-specific insertion that follows the furin-like processing site, suggesting KDa that interactions mediated by this region are likely independent 100 of furin-like processing and this insertion. Glypican mutations 75 previously associated with functional impairments, for example those in glypican-3 that cause Simpson–Gohlabi–Behmel syn- 50 drome (42, 43) and those in glypican-6 that cause omodysplasia 37 (7), appear to result in severe truncations or complete loss of expression of the affected glypican. Whether the C-lobe region 25 we have identified on Dlp is generally involved in glypican func- 20 tion or is specific to Dlp or positive regulators of Hh signaling is 15 an interesting question for future investigation. The results pre- 10 sented here establish a molecular foundation to guide design and interpretation of studies investigating the molecular bases of Fig. 4. Interactions between Hh and glypicans. (A) Coomassie brilliant blue– stained SDS-PAGE analysis of pull-down assays in which Drosophila HhN was glypican function. immobilized on Ni-NTA agarose and incubated with the indicated partners. Red arrows indicate bands corresponding to input proteins in control lanes. Materials and Methods Each glypican consists of a larger N-terminal domain and smaller C-terminal Dlp Expression and Purification. Amino acid sequence alignments and predic- domain in reducing gels. (B) Coomassie briliant blue-stained SDS-PAGE tions of both the site of signal sequence cleavage and positions of secondary analysis of pull-down assays in which murine ShhN was immobilized on structural elements (4, 44) led to identification of Dlp residues 60–617

Ni-NTA agarose and incubated with the indicated partners (Gpc6, glypi- (numbering from the initiator methionine) as the core folded region of BIOPHYSICS AND Dlp. A gene encoding this region of Dlp was subcloned into the pSGHV0 can-6 N-terminal protein core; Gpc3, glypican-3 N-terminal protein core). COMPUTATIONAL BIOLOGY −∕− ShhN runs just above the 25-kDa marker. expression vector (45) and transfected into dhfr CHO cells (29). Purifica- tion of this fragment showed it to undergo partial proteolysis at its N terminus and partial processing at both consensus and cryptic furin-recognition One reason Dlp has been proposed as an Hh coreceptor is the sequences at residues 397 and 437, respectively. To generate a more homo- ability of Dlp lacking heparan sulfate to coimmunoprecipitate genous form of Dlp (DlpΔNCF), the gene encoding a Dlp fragment spanning with Hh (26). Our inability to detect a high-affinity interaction residues 74–617 but lacking residues 400–437, which intervene between the between DlpΔNCF and HhN using purified proteins suggests that two sites of partial proteolytic processing, and with Asn 79 and Asn 502 sub- Dlp may interact with Hh as part of a larger complex and addi- stituted with glutamate to remove the two consensus N-linked glycosylation tional factors are needed to promote Dlp∕Hh interactions (25). attachments, was subcloned into pSGHV0 and stably tranfected into dhfr−∕− One candidate for such a factor is the Hh coreceptor Ihog, which CHO cells. DlpΔNCF expression levels were amplified by selection of cell lines in is an essential component of the Hh receptor complex (33), but methotrexate (46), and DlpΔNCF purified from conditioned medium using immobilized metal ion affinity, anion exchange, and size-exclusion chroma- we show here that purified DlpΔNCF does not form a high-affinity – tographies. Final yields of purified DlpΔNCF were 1 2 mg per liter of condi- complex with either an active fragment of Ihog (IhogFn12) or an tioned medium.

HhN:IhogFn12 complex. This result does not rule out Ihog as To prepare selenomethionine (SeMet)-labeled DlpΔNCF, cells were washed important for mediating Hh:Dlp interactions but suggests that once with Hanks’ balanced salt solution and incubated in ExCell301 medium an additional factor or factors may be needed. An obvious can- (JRH Biosciences) lacking methionine but supplemented with 50 μg∕mL didate for such a factor is Patched, a key cell-surface component L-SeMet (Sigma). To optimize incorporation of SeMet into DlpΔNCF, the first of the Hh signaling pathway, but assessing its role in Hh-contain- exchange of SeMet-containing media was removed after 24 h and discarded. ing complexes awaits purification of suitable amounts of this DlpΔNCF was purified from later collections of conditioned media. 12-pass integral membrane protein. An additional element com- – Dlp Crystallization and Structure Determination. Drosophila DlpΔNCF was crys- plicating interpretation of results of studies of receptor ligand tallized by the hanging drop vapor diffusion method. One microliter of a interactions in solution arises from the absence of membrane 3 5 ∕ . mg mL solution of DlpΔNCF was mixed with an equal volume of a reservoir tethering and ligand multivalency. Restricting components to a solution containing 0.1 M MES pH 6.7, 0.2 M Mg formate and 20% PEG3350. membrane surface orients them and greatly enhances their local Crystals grew to final dimensions of 0.2 × 0.2 × 0.05 mm3 after 1 week at

Kim et al. PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32 ∣ 13115 Downloaded by guest on September 30, 2021 20 °C. DlpΔNCF crystals were briefly soaked in crystallization buffer containing attachment sites in GPC6. Expression and purification of GPC3 and GPC6 pro- ∕ 10% vol vol PEG 200 prior to freezing in liquid nitrogen for X-ray data tein domains were carried out similarly to DlpΔNCF. Final yields of purified collection. DlpΔNCF crystals belong to space group C2 with unit cell dimensions GPC3 and GPC6 were 0.5 mg and 1 mg per liter of conditioned medium, a ¼ 97.02, b ¼ 66.42, c ¼ 85.73 Å, and β ¼ 104.85°. X-ray diffraction data respectively, and the purified proteins showed almost complete furin proces- were collected from SeMet-substituted DlpΔNCF crystals at selenium peak, sing. DmHh, IhogFN12, ShhN, and CDOFn3 were purified as previously edge and remote wavelengths, and from native crystals at the peak wave- described. Briefly, DNA fragments encoding mouse ShhH (26–189) and length at the Lilly Research Laboratories Collaborative Access Team beam line human CDOFn3 (826–924) were cloned into the bacterial expression plasmid at the Advance Photon Source at Argonne National Laboratory. Diffraction pT7HMT (54). DNA fragments encoding DmHh and IhogFn12 were cloned data were processed using the program HKL2000 (47), and the program into a modified pMAL-c2X (NEB) bacterial expression vector. Proteins were SOLVE/RESOLVE was used to find 14 out of 17 selenium sites and calculate expressed in the BL21(DE3) Escherichia coli strain and purified using immo- initial phases (48). The program COOT was used for model building (49), bilized metal ion affinity followed by digestion with tobacco etch virus and refinement was performed with the programs PHENIX (50) and BUSTER protease to remove N-terminal tags. Proteins were further purified by anion – – – – (51). The final model consists of residues 120 288, 293 393, 509 569, 572 exchange and size-exclusion chromatographies. 576, and 589–614 and 69 water molecules. Weak electron density is present for unmodeled regions in the N-terminal lobe, but extensive effort failed to Pull-Down Binding Assays. Eight histidine-tagged ShhN protein was generate an acceptable model for this region. adsorbed to Ni-NTA resin (GE Healthcare) for 1 h at room temperature with gentle rocking. The ShhN-loaded resin was washed three times with binding Dlp Activity Assay. Mutations were introduced into the gene encoding buffer (20 mM Tris pH 8.0, 0.2 M NaCl, 20 mM imidazole, and 1 mM CaCl2) Dlp∆GAG by the megaprimer method (52). The ability of Dlp variants to med- and incubated with combinations of 26 μM glypican-6 N-terminal domain, iate Hh responsiveness in clone-8 cells using a luciferase-reporter assay was glypican-3 N-terminal domain, and CDOFn3 for 1 h at 20 °C. The resin was assayed as described previously (25). washed three times, boiled in SDS-loading buffer, and analyzed by SDS-PAGE using Coomassie brilliant blue staining. Cloning, Expression, and Purification of GPC3, GPC6 and CDO Fragments. DNA fragments encoding the human GPC3 N-terminal domain (residues 32–480) and mouse GPC6 N-terminal domain (residues 24–480) were amplified by ACKNOWLEDGMENTS. We thank Stephen Wasserman of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) for assistance with X-ray PCR, cloned into the pSGHV0 vector (45), and expressed in CHO cells. Purified data collection. This work was supported by the Howard Hughes Medical GPC3 and GPC6 showed partial processing at cryptic furin-recognition Institute (P.A.B.) and the National Institutes of Health (Grant R01HD055545 sequences. To increase the furin processing efficiency of both GPC3 and to D.J.L.). Use of the Advanced Photon Source at Argonne National Labora- – GPC6, the furin-recognition sites of GPC3 (355 358; RQYR) and GPC6 tory was supported by the US Department of Energy, Office of Science, Office (351–354; RSAR) were changed to RRRRRR using megaprimer PCR mutagen- of Basic Energy Sciences, under Contract DE-AC02-06CH11357. Use of the esis (53). The asparagines of the two N-linked glycosylation attachment sites LRL-CAT beamline at sector 31 of the Advanced Photon Source was provided in GPC3 (N124, N418) were substituted with glutamate; there are no N-linked by Eli Lilly Company, which operates the facility.

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