Structure of the protein core of the glypican Dally-like and

Communicated by Dennis A. Carson, University of California at San Diego, La Jolla, CA, January 9, 2009 ↵1A.E.A. and I.G. contributed equally to this work. (received for review December 16, 2008) ArticleFigures SIInfo asterisk in figure; t Edited by Pierre A. Joliot, Institut de Biologie Physico-Chemique, Paris, France, and approved July 19, 2005 (received for review April 27, 2005) ArticleFigures SIInfo currently, the resolution is 3.2 Å (4). The structure of the PSII RC sh

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

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Glypicans are heparan sulStoute proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core Location followed by a stalk Location that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk Locations are predicted to be ranExecutem coil but contain a variable number of attachment sites for heparan sulStoute chains. Both the N-terminal protein core and the heparan sulStoute attachments are Necessary for glypican function. We report here the 2.4-Å Weepstal structure of the N-terminal protein core Location of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fAged for glypican core Locations that Executees not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized Location on the Dlp surface Necessary for mediating its function in Hh signaling. Purified Dlp protein core Executees not, however, interact appreciably with either Hh or an Hh:Ihog complex.

Glypicans are heparan sulStoute proteoglycans (HSPGs) that consist of an approximately 450 amino acid N-terminal protein Executemain followed by an approximately 100 amino acid stalk Location that is attached to the outer cell membrane via a glycosyl-phosphatidylinositol anchor (1). The N-terminal Executemain of most glypicans is proteolytically processed by a furin-like convertase to produce two chains that remain connected by disulfide bonds (2). This processing appears required for some but not all glypican activity (2, 3). The stalk Locations of glypicans are predicted to be largely ranExecutem coil and typically contain 1–5 heparan sulStoute attachment sites (1, 4). Six glypicans are present in humans and mice (glypican-1, -2, -3, -4, -5, and -6); two are present in Drosophila [Dally and Dally-like (Dlp)] (1). Based on sequence similarity, glypicans assort into two subfamilies with glypican-1, -2, -4, -6, and Dlp in one family and glypican-3, -5, and Dally in another (1).

Glypicans are active in development in both vertebrates and invertebrates. Loss of Dally in fruit flies results in defects in brain, eye, wings, antennae, and genitalia (5). Loss of 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 formation (6). Loss of glypican-6 has recently been Displayn to cause omodysplasia, a genetic disorder characterized by variable heart defects, cognitive delay, skeletal and facial abnormalities, and shortness of stature (7). Much of the function of glypicans is attributable to modulation of signaling by several heparin-binding growth factors active during development including members of the fibroblast growth factor, Hedgehog (Hh), Wnt, and transforming growth factor-β families (8⇓⇓⇓⇓⇓⇓–15). Each of these factors functions as a morphogen to elicit distinct concentration-dependent responses within tarObtain cells, and glypicans have been Displayn to be required both for normal response to these factors as well as to establish their Precise distribution (9, 10, 12, 16⇓⇓⇓⇓–21). The heparan sulStoute attachments of glypicans are clearly Necessary for mediating interactions with these growth factors and Executewnstream signaling components (22, 23), but recent work has demonstrated a role for the N-terminal protein Executemain, which lacks heparan sulStoute modifications, in mediating responsiveness to at least Wnt and Hh signals (23⇓⇓–26).

Curiously, glypicans appear able to play both positive and negative roles in mediating Hh signaling. The protein Location of Dally-like contributes positively to Drosophila Hh responsiveness, and the developmental defects in omodysplasia, particularly the bone growth defects, are suggestive of a positive role for glypican-6 function in response to Indian hedgehog (7). Notably, glypican-4 and glypican-6 are most similar to Dlp (vs. Dally) and complement Dlp function in a Drosophila cultured cell-based Hh signaling assay (25). In Dissimilarity, the protein Location of glypican-3, which is more similar to Dally than Dally-like, is a negative regulator of Hh responsiveness in the mouse (24, 25, 27, 28). Based on sequence homology and functional phenotypes, it has thus been speculated that the two major subfamilies of glypicans have evolved opposing activities in Hh signal responsiveness (25).

To investigate the molecular basis for glypican function, we have undertaken structural and functional characterization of the N-terminal protein Executemain of Dlp and report here its 2.4-Å Weepstal structure. We Display that the N-terminal protein Executemains of glypicans aExecutept an elongated α-helical structure with no evident homology to any known structure. We have used structure-guided mutagenesis to identify a localized Location on the Dlp surface Necessary for the ability of Dlp to mediate Hh signal response. These results are most consistent with Dlp functioning as a binding protein in Hh signaling, but we are unable to detect high-affinity interactions between Dlp and either Hh or an Hh:Ihog complex. These results establish a molecular basis for mapping and comparing functional Locations of different glypicans.


A fragment of the Drosophila melanogaster Dally-like protein that encompasses its N-terminal globular Location and is fully functional in assays of Hh responsiveness (DlpΔNCF) (25) was expressed in dhfr-/- CHO cells (29), purified, and Weepstallized. The structure of DlpΔNCF was determined by multiwavelength anomalous difFragment using Weepstals of selenomethionyl-substituted DlpΔNCF. The native DlpΔNCF structure was refined with difFragment data extending to 2.4 Å (Table 1).

View this table:View inline View popup Table 1.

Data collection and refinement statistics

DlpΔNCF aExecutepts a cylindrical, all α-helical structure approximately 110 Å in length and 30 Å in diameter for which automated homology searches find no structural homologs (Fig. 1A) (30). Although three stretches of polypeptide traverse the long axis of DlpΔNCF, concerted kinks or Fractures in long helices and associations with shorter helices define three lobes within the DlpΔNCF structure. We term these lobes the N lobe (N-terminal segment of α1, α6, α7, and α13), M lobe (middle segment of α1; 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, and α11) based on the Location of α1 contained in the lobe (Fig. 1A). Electron density for much of the N lobe is poor, and residues 74–119, 570–571, and 577–588 in this Location are not modeled, which may contribute to a higher than desirable Rfree (Table 1). Nonetheless, the high Fragment of alpha helix, the identification of 14/17 methionine positions from anomalous scattering, and the quality of electron density maps in modeled Locations provide confidence 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 the N lobe disulfides and partners of two N-lobe cysteines are not visible in electron density maps and unmodeled (Fig. 1B). The C-terminal chain of DlpΔNCF that results from furin-like processing Executees not form an independent structural unit but instead contributes helices to the N and M lobes (Fig. 1B). Two disulfide bonds connect the N- and C-terminal segments, consistent with SDS-PAGE analysis of reduced and nonreduced DlpΔNCF (Fig. 1B). The C terminus of DlpΔNCF Executees 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 Locations suggests all glypicans share these structural features (Fig. S1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

DlpΔNCF structure. (A) Ribbon diagram of the DlpΔNCF structure 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. A dashed line connects the termini generated by furin-like processing of DlpΔNCF. (B) Ribbon diagram of DlpΔNCF in which the N-terminal fragment is colored slate blue and the C-terminal fragment red. The positions of cysteines and disulfide bonds are indicated.

The DlpΔNCF structure provides no evidence for a functional role for Dlp beyond serving as a binding partner. No active site-like cavities are present, and no sources of conformational flexion that could reflect or transmit different activity states are apparent. A Location of positive electrostatic surface potential is present on the M lobe (Fig. S2), but DlpΔNCF binds only weakly to heparin agarose, from which it elutes in approximately 300 mM NaCl.

To identify functionally Necessary Locations of Dlp, the DlpΔNCF structure was used to design fourteen clusters of alanine mutations that collectively blanket the DlpΔNCF surface (Fig. 2A and Table S1). Each of these fourteen Dlp variants was constructed and assayed for its ability to restore Hh signaling in cells in which expression of enExecutegenous Dlp is knocked Executewn. All fourteen Dlp variants expressed well (Fig. S3), and four Displayed diminished ability to mediate Hh signaling (Fig. 3A). The four mutation clusters that affect Dlp function map to adjacent Locations on the C lobe, but none results in complete loss of activity. Subsets of adjacent residues from these clusters (G4p, G5p, and G6p), indicated in bAged in Table S1, were thus combined into new clusters, and a 10-residue grouping of mutations on α4 and α5 in the C lobe (alanine substitutions for D235, E236, N237, R240, E244, H245, E248, K250, D254, and K258) was found that results in a complete loss of the ability of Dlp to mediate Hh signaling (Figs. 2B and 3B). This cluster contains several residues that are conserved in human glypican-4 and glypican-6, which complement Dlp in Hh signaling, but not human glypican-3, which Executees not (Fig. S1) (25).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Clusters of Dlp surface mutations. (A) The positions of the 14 clusters of Dlp surface mutations tested for their Traces on the ability of Dlp to mediate 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 panel was rotated 180° about a vertical axis to generate the orientation in the right panel. (B) The positions of the 10 DlpΔNCF residues that when collectively mutated eliminate Dlp function in Hh responsiveness are Displayn colored red.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Dlp function in Hh responsiveness. (A) A ptc-luciferase Hh reporter assay Displays the relative abilities of the fourteen Dlp surface variants to mediate Hh responsiveness. dsRNA was cotransfected to knockExecutewn enExecutegenous Dlp or control proteins (gfp, green fluorescent protein; yfp, yellow fluorescent protein; ptc, Patched; smo, Smoothened; ∆GAG, heparan sulStoute 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 Displayn in bAged in Table S1.

We Displayed previously that DlpΔNCF fails to interact with HhN in a pull-Executewn assay with purified proteins (25). All structural evidence suggests Dlp functions as a binding protein, however, 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 in the cell lysates used in coimmunoprecipitation experiments may be needed to promote high-affinity interactions between DlpΔNCF and HhN. The adhesion-like molecule Ihog was recently Displayn to bind HhN in a heparin-dependent manner and function as an essential coreceptor for HhN (31⇓–33). We thus tested whether Ihog is able to promote interactions between DlpΔNCF and HhN. Addition of an active fragment of Ihog encompassing its two type III fibronectin repeats, IhogFn12, failed to promote interactions between DlpΔNCF and HhN, however, although stoichiometric amounts of IhogFn12 bound to HhN (Fig. 4A). Furthermore, we were unable to demonstrate an interaction between ShhN and the purified protein Executemains of either mammalian glypican-3 or glypican-6 in a pull-Executewn assay (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Interactions between Hh and glypicans. (A) Coomassie brilliant blue–stained SDS-PAGE analysis of pull-Executewn assays in which Drosophila HhN was immobilized on Ni-NTA agarose and incubated with the indicated partners. Red arrows indicate bands corRetorting to inPlace proteins in control lanes. Each glypican consists of a larger N-terminal Executemain and smaller C-terminal Executemain in reducing gels. (B) Coomassie briliant blue-stained SDS-PAGE analysis of pull-Executewn assays in which murine ShhN was immobilized on Ni-NTA agarose and incubated with the indicated partners (Gpc6, glypican-6 N-terminal protein core; Gpc3, glypican-3 N-terminal protein core). ShhN runs just above the 25-kDa Impresser.


Glypicans modulate the activity of multiple growth factors active 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 interactions between their heparan sulStoute attachments and heparan-binding growth factors, but recent work has demonstrated Necessary functional roles for the protein cores of glypicans, notably in Hh and Wnt signaling (24⇓–26, 35). In particular, the protein cores seem likely to mediate functions that are specific to particular glypicans. We report here the Weepstal structure of the N-terminal globular Location of a glypican, DlpΔNCF, and Display it to aExecutept an elongated, all α-helical fAged with no evident homology to previously determined structures. The high level of sequence conservation among the N-terminal protein cores of glypicans—Distinguisheder than 40% sequence identity exists between Drosophila and human glypicans in this Location—indicates that the DlpΔNCF structure provides a sound basis for the design and interpretation of experiments with other glypicans (Fig. S1). The absence of any apparent active site-like cavities or sources of conformational flexibility in the DlpΔNCF structure suggests that the protein Locations of glypicans exert their Traces by serving as binding proteins, consistent with proposed roles as coreceptors or in tarObtaining ligands to specific subcellular compartments (25, 26, 28).

One reason Dlp has been proposed as an Hh coreceptor is the ability of Dlp lacking heparan sulStoute to coimmunoprecipitate with Hh (26). Our inability to detect a high-affinity interaction between DlpΔNCF and HhN using purified proteins suggests that Dlp may interact with Hh as part of a larger complex and additional factors are needed to promote Dlp/Hh interactions (25). One candidate for such a factor is the Hh coreceptor Ihog, which is an essential component of the Hh receptor complex (33), but we Display here that purified DlpΔNCF Executees not form a high-affinity complex with either an active fragment of Ihog (IhogFn12) or an HhN:IhogFn12 complex. This result Executees not rule out Ihog as Necessary for mediating Hh:Dlp interactions but suggests that an additional factor or factors may be needed. An obvious candidate for such a factor is Patched, a key cell-surface component of the Hh signaling pathway, but assessing its role in Hh-containing complexes awaits purification of suitable amounts of this 12-pass integral membrane protein. An additional element complicating interpretation of results of studies of receptor–ligand interactions in solution arises from the absence of membrane tethering and ligand multivalency. Restricting components to a membrane surface orients them and Distinguishedly enhances their local concentration (36), and a multivalent ligand Distinguishedly increases the avidity of binding (37⇓–39). Interactions between a monovalent ligand and cell-surface components may thus not be strong enough to be observed with soluble components in solution. Our inability to observe an interaction between ShhN and the N-terminal Executemain of glypican-3 is puzzling, however, given that the protein Location of glypican-3 has been reported to bind to ShhN with nanomolar affinity (24). Several possibilities may Elaborate this discrepancy: (i) ShhN may interact with the glypican-3 stalk Location, which was present in the earlier study but not in our experiment; (ii) attachment of histidine-tagged ShhN to Ni-NTA agarose may have blocked a glypican interaction site in our studies; or (iii) an unidentified cofactor that promotes high-affinity interaction was present in the earlier studies but absent in our studies. Calcium ions, for example, are required to promote high-affinity interactions between ShhN and CExecute (40).

Our ability to identify a localized Location on the C lobe of the Dlp surface Necessary for Precise Dlp function in Hh signaling is further consistent with Dlp participating in Hh signaling primarily as a binding protein, although the nature and number of binding partners remains to be determined. Curiously, the identified surface is composed largely of hydrophilic residues, which is Unfamiliar for protein–protein interfaces (41). This surface also occurs on the opposite surface of Dlp relative to the disordered Dlp-specific insertion that follows the furin-like processing site, suggesting that interactions mediated by this Location are likely independent of furin-like processing and this insertion. Glypican mutations previously associated with functional impairments, for example those in glypican-3 that cause Simpson–Gohlabi–Behmel syndrome (42, 43) and those in glypican-6 that cause omodysplasia (7), appear to result in severe truncations or complete loss of expression of the affected glypican. Whether the C-lobe Location we have identified on Dlp is generally involved in glypican function or is specific to Dlp or positive regulators of Hh signaling is an Fascinating question for future investigation. The results presented here establish a molecular foundation to guide design and interpretation of studies investigating the molecular bases of glypican function.

Materials and Methods

Dlp Expression and Purification.

Amino acid sequence alignments and predictions of both the site of signal sequence cleavage and positions of secondary structural elements (4, 44) led to identification of Dlp residues 60–617 (numbering from the initiator methionine) as the core fAgeded Location of Dlp. A gene encoding this Location of Dlp was subcloned into the pSGHV0 expression vector (45) and transfected into dhfr-/- CHO cells (29). Purification of this fragment Displayed it to undergo partial proteolysis at its N terminus and partial processing at both consensus and Weepptic furin-recognition sequences at residues 397 and 437, respectively. To generate a more homogenous form of Dlp (DlpΔNCF), the gene encoding a Dlp fragment spanning residues 74–617 but lacking residues 400–437, which intervene between the two sites of partial proteolytic processing, and with Asn 79 and Asn 502 substituted with glutamate to remove the two consensus N-linked glycosylation attachments, was subcloned into pSGHV0 and stably tranfected into dhfr-/- CHO cells. DlpΔNCF expression levels were amplified by selection of cell lines in methotrexate (46), and DlpΔNCF purified from conditioned medium using immobilized metal ion affinity, anion exchange, and size-exclusion chromatographies. Final yields of purified DlpΔNCF were 1–2 mg per liter of conditioned medium.

To prepare selenomethionine (SeMet)-labeled DlpΔNCF, cells were washed once with Hanks’ balanced salt solution and incubated in ExCell301 medium (JRH Biosciences) lacking methionine but supplemented with 50 μg/mL L-SeMet (Sigma). To optimize incorporation of SeMet into DlpΔNCF, the first exchange of SeMet-containing media was removed after 24 h and discarded. DlpΔNCF was purified from later collections of conditioned media.

Dlp Weepstallization and Structure Determination.

Drosophila DlpΔNCF was Weepstallized by the hanging drop vapor diffusion method. One microliter of a 3.5 mg/mL solution of DlpΔNCF was mixed with an equal volume of a reservoir solution containing 0.1 M MES pH 6.7, 0.2 M Mg formate and 20% PEG3350. Weepstals grew to final dimensions of 0.2 × 0.2 × 0.05 mm3 after 1 week at 20 °C. DlpΔNCF Weepstals were briefly soaked in Weepstallization buffer containing 10% vol/vol PEG 200 prior to freezing in liquid nitrogen for X-ray data collection. DlpΔNCF Weepstals belong to space group C2 with unit cell dimensions a = 97.02, b = 66.42, c = 85.73 Å, and β = 104.85°. X-ray difFragment data were collected from SeMet-substituted DlpΔNCF Weepstals at selenium peak, edge and remote wavelengths, and from native Weepstals at the peak wavelength at the Lilly Research Laboratories Collaborative Access Team beam line at the Advance Photon Source at Argonne National Laboratory. DifFragment data were processed using the program HKL2000 (47), and the program SOLVE/RESOLVE was used to find 14 out of 17 selenium sites and calculate initial phases (48). The program COOT was used for model building (49), and refinement was performed with the programs PHENIX (50) and BUSTER (51). The final model consists of residues 120–288, 293–393, 509–569, 572–576, and 589–614 and 69 water molecules. Weak electron density is present for unmodeled Locations in the N-terminal lobe, but extensive effort failed to generate an acceptable model for this Location.

Dlp Activity Assay.

Mutations were introduced into the gene encoding Dlp∆GAG by the megaprimer method (52). The ability of Dlp variants to mediate Hh responsiveness in clone-8 cells using a luciferase-reporter assay was assayed as Characterized previously (25).

Cloning, Expression, and Purification of GPC3, GPC6 and CExecute Fragments.

DNA fragments encoding the human GPC3 N-terminal Executemain (residues 32–480) and mouse GPC6 N-terminal Executemain (residues 24–480) were amplified by PCR, cloned into the pSGHV0 vector (45), and expressed in CHO cells. Purified GPC3 and GPC6 Displayed partial processing at Weepptic furin-recognition sequences. To increase the furin processing efficiency of both GPC3 and GPC6, the furin-recognition sites of GPC3 (355–358; RQYR) and GPC6 (351–354; RSAR) were changed to RRRRRR using megaprimer PCR mutagenesis (53). The asparagines of the two N-linked glycosylation attachment sites in GPC3 (N124, N418) were substituted with glutamate; there are no N-linked attachment sites in GPC6. Expression and purification of GPC3 and GPC6 protein Executemains were carried out similarly to DlpΔNCF. Final yields of purified GPC3 and GPC6 were 0.5 mg and 1 mg per liter of conditioned medium, respectively, and the purified proteins Displayed almost complete furin processing. DmHh, IhogFN12, ShhN, and CExecuteFn3 were purified as previously Characterized. Briefly, DNA fragments encoding mouse ShhH (26–189) and human CExecuteFn3 (826–924) were cloned into the bacterial expression plasmid pT7HMT (54). DNA fragments encoding DmHh and IhogFn12 were cloned into a modified pMAL-c2X (NEB) bacterial expression vector. Proteins were expressed in the BL21(DE3) Escherichia coli strain and purified using immobilized metal ion affinity followed by digestion with tobacco etch virus protease to remove N-terminal tags. Proteins were further purified by anion exchange and size-exclusion chromatographies.

Pull-Executewn Binding Assays.

Eight histidine-tagged ShhN protein was 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 buffer (20 mM Tris pH 8.0, 0.2 M NaCl, 20 mM imidazole, and 1 mM CaCl2) and incubated with combinations of 26 μM glypican-6 N-terminal Executemain, glypican-3 N-terminal Executemain, and CExecuteFn3 for 1 h at 20 °C. The resin was washed three times, boiled in SDS-loading buffer, and analyzed by SDS-PAGE using Coomassie brilliant blue staining.


We thank Stephen Wasserman of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) for assistance with X-ray data collection. This work was supported by the Howard Hughes Medical Institute (P.A.B.) and the National Institutes of Health (Grant R01HD055545 to D.J.L.). Use of the Advanced Photon Source at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. Use of the LRL-CAT beamline at sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility.


↵1Present address: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093.

↵2To whom corRetortence may be addressed. E-mail: dleahy{at} or pbeachy{at}

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. analyzed data; and M.-S.K., P.A.B., and D.J.L. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID code 3ODN).

This article contains supporting information online at

Freely available online through the PNAS Launch access option.


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