Fj

RRKRR

cc cc cc

II III I I

MMMM

Brachydactyly type Al Brachydactyly type A2 Brachydactyly type C

Du Pan syndrome Grebe syndrome Hunter-Thompson syndrome*

Osteoarthritis susceptibility proximal Symphalangism 1 Multiple synostosis syndrome 1 Multiple synostosis syndrome 2**

Figure 8. Localization of GDF5 mutations. Arrowheads indicate the location of all currently known mutations linked to human skeletal malformation diseases affecting the limb. The specific inherited disease caused by each mutation is displayed in the legend underneath.

A GDF-5 monomer consists of an N-terminal signal peptide domain (black box), a prodomain (dark grey box) and the C-terminal mature part (light grey box) containing six highly conserved cysteine residues forming the cystine knot motif, whereas the seventh cysteine connects two monomers via an intermolecular disulfide bond. Italic type indicates nucleotide nomenclature; normal type represents single amino acid nomenclature. For references see Table 1.

mutation

location

hetero-/homozygous

disease

OMIM #

reference

rs143383

5'UTR gdf5 gene

heterozygous

Osteoarthritis susceptibility

#612400

(Miyamoto et al., 2007)

rs143384

5'UTR gdf5 gene

heterozygous

Osteoarthritis susceptibility

#612400

(Rouault et al, 2010)

2250ct

3'UTR gdf5 gene

heterozygous

Osteoarthritis susceptibility

#612400

(Egli et al., 2009)

121delG

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Polinkovsky et al, 1997)

158delT

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Everman et al, 2002)

158insC

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Everman et al, 2002)

206insG

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Polinkovsky et al, 1997)

206insG

prodomain gdf5 gene

homozygous

Chondrodysplasia, Grebe type

#200700

(Stelzer et al., 2003)

297insC

prodomain gdf5 gene

homozygous

Chondrodysplasia, Grebe type

# 200700

(Faiyaz-Ul-Haque et al., 2002a)

493delC

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Galjaard et al, 2001)

M173 V

prodomain gdf5 gene

homozygous

Brachydactyly type C

#113100

(Schwabe et al, 2004)

S204R

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Everman et al, 2002)

759delG

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Polinkovsky et al, 1997)

811ins23

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

Everman, D. B. et al. 2002)

830delT

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

Everman, D. B. et al. 2002)

R301X

prodomain gdf5 gene

heterozygous

Brachydactyly type C

#113100

(Polinkovsky et al, 1997)

1114insGAGT

prodomain gdf5 gene

homozygous

Chondrodysplasia, Grebe type

#200700

(Basit et al., 2008)

R378Q/P436T

prodomain gdf5 gene; processing site / mature domain

compound heterozygous

Acromesomelic dysplasia, DuPan syndrome

#601146

(Douzgou et al, 2008)

R380Q

prodomain gdf5 gene; processing site

heterozygous

Brachydactyly type A2

#112600

(Ploger et al., 2008)

R399C

mature domain

heterozygous

Brachydactyly type A1

#112500

(Byrnes et al., 2010)

C400Y

mature domain; no processing/secretion

heterozygous

Brachydactyly type C

#113100

(Thomas et al, 1997)

C400Y

mature domain; no processing/secretion

homozygous

Chondrodysplasia, Grebe type

#200700

(Thomas et al, 1997)

C400Y/del1144G

mature domain/

compound

Chondrodysplasia,

#200700

(Thomas et

prodomain; no processing/secretion

heterozygous

Grebe type

al., 1997)

mW408R (hW414R)

mature domain; location in type I receptor binding site

heterozygous

Brachypodism

(Masuya et al, 2007)

mW408R (hW414R)

mature domain; location in type I receptor binding site

homozygous

severe Brachypodism, Osteoarthritis

(Masuya et al, 2007)

C429R

mature domain

homozygous

Chondrodysplasia, Grebe type

#200700

(Faiyaz-Ul-Haque et al., 2008)

Table 1. Table of all known mutations in GDF5 gene linked to skeletal malformation diseases affecting the limb. Mutations depicted in red represent single nuclear polymorphisms (SNPs) located in 5' or 3' regulatory regions of GDF5 gene. Shown in black are mutations situated in the prodomain, whereas mutations in the mature part are represented in blue. Frameshift mutations are highlighted in italics, non-sense mutations are underlined.

Table 1. Table of all known mutations in GDF5 gene linked to skeletal malformation diseases affecting the limb. Mutations depicted in red represent single nuclear polymorphisms (SNPs) located in 5' or 3' regulatory regions of GDF5 gene. Shown in black are mutations situated in the prodomain, whereas mutations in the mature part are represented in blue. Frameshift mutations are highlighted in italics, non-sense mutations are underlined.

polymorphism is not restricted to cartilage (Egli et al., 2009). In addition, recent analysis showed that expression of GDF-5 could be further modulated epigenetically as both Calleles of the SNPs rs143383 and rs143384 form CpG sites thereby explaining the intra- and inter-individual variations observed (Reynard et al., 2011). A third SNP influencing GDF-5 expression, 2250ct, is found in the 3' UTR of GDF5. It acts independently from the 5' SNP rs143383 and can similarly reduce protein expression levels by 20-25% (Egli et al., 2009). The independent reduction in expression by these SNPs can be additive thereby showing that even moderate imbalances in the allelic expression levels of GDF5 can result in severe disturbances in synovial joint maintenance. This idea is further emphasized by the identification of a duplication in the 3' UTR of the BMP2 gene including a distant enhancer of BMP2 expression in BDA2 patients. The phenotype described by Dathe et al. resembles those caused by specific mutations in the GDF5 or the BMPR1B gene (Dathe et al., 2009). As BMP-2 is expressed in regions surrounding future joints as well as in the joint interzone during the development of interphalangeal joints in close proximity to GDF-5 expression, one could hypothesize that by either increasing BMP-2 levels due to the duplication of an enhancer or by decreasing the GDF-5 expression due to regulatory SNPs as described above, the fine-tuned balance between signals from different BMPs may be severely disturbed.

3.5. Proper folding and processing of pro-GDF-5 is essential for GDF-5 signaling

Like other ligands of the TGF-P superfamily GDF-5 is expressed and secreted as a dimeric pro-protein consisting of a large (354aa per monomer) pro-part and a smaller (120aa per monomer) mature part at the C-terminus. The C-terminal mature part harbors the characteristic motif present in all TGF-P ligands comprising of seven (BMPs, GDFs) highly conserved cysteine residues (Activins, TGF-Ps have two further Cys residues at the N-terminus of the mature part) of which six form the so-called cystine knot. The seventh cysteine residue is involved in an intermolecular disulfide bond thereby stabilizing the (usually homo-)dimeric ligand assembly. The dimeric mature part of TGF-P ligand exhibits a butterfly shaped assembly with the monomeric subunits adopting an architecture resembling a left hand (Sebald et al., 2004). The dimer interface is formed by the palm of the hand, two two-stranded P-sheets resembling two fingers emanate from the cystine knot containing palm. Mutagenesis was used to determine the receptor binding epitopes (Kirsch et al., 2000). The BMP type I receptors bind to the so-called wrist epitope, the type II receptors bind to the so-called knuckle epitope (Kirsch et al., 2000). The location of these receptor binding epitopes were then confirmed by structure analyses of various BMP ligand-receptor complexes (Kirsch et al., 2000, Greenwald et al., 2003, Allendorph et al., 2006, Weber et al., 2007, Kotzsch et al., 2009).

Homozygous non-sense or frame-shift mutations in the pro- or mature part of GDF5 will result in a complete knockout of GDF5. However, also heterozygous non-sense and frame-shift mutations in GDF5 will severely lower the level of intact protein; assuming equal transcriptional and translational efficiency from both alleles by statistics only 25% of the protein produced will be intact due to its dimeric nature. Hence the complete knockout or partial knockdown of GDF5 achieved by this type of mutation leads to rather severe skeletal malformation phenotypes such as brachydactyly type C (BDC), symphalangism (SYM1) or multiple synostosis syndrome (SYNS1). One potentially underappreciated possibility is also the formation of nonfunctional heterodimeric ligands if a cell produces more than one TGF-P factor at a time and thus a possible influence of non-sense GDF-5 mutations onto other BMP signals. It is a known fact that in Drosophila the BMP-2 and BMP-7 orthologs Dpp and Screw can form heterodimers with unique functions required for proper development of certain tissues (Shimmi et al., 2005, O'Connor et al., 2006), however in vertebrates existence of such BMP heterodimers has only been postulated or recombinant proteins have been used in the analysis, but existence of such heterodimers has not really been proven in vivo (Schmid et al., 2000, Butler & Dodd, 2003) thus a potential "cross"-influence of nonfunctional GDF-5 mutations on other BMPs can only be hypothesized.

Of the 14 missense mutations known in the GDF5 gene four are located within the pro-part of the GDF-5 protein. Whereas for the TGF-Ps the pro-part fulfills an important regulatory role, termed latency, its role for the BMP and GDF subgroup of the TGF-P superfamily is much less clear. Latency was discovered for TGF-P1 in 1984 showing that TGF-P proteins are secreted as large protein complexes that require activation for TGF-P signaling (Lawrence et al., 1984). It is known today that upon secretion the pro-part of TGF-Ps is cleaved in the Golgi apparatus by furin proteases at a site between the pro- and mature part containing a consensus RXXR motif (other proteases might substitute for furin proteases but providing for TGF-P proteins with different N-termini) (Dubois et al., 1995). The pro-part also called latency-associated peptide (LAP) however is still non-covalently attached thereby interfering with TGF-P signaling. Activation corresponding to release of the mature part from this intermediate latent complex is achieved either by physicochemical changes in the environment, e.g. acidification or by further proteolysis. Proteins specifically binding LAP have been identified (Miyazono et al., 1988), these latent TGF-P binding proteins (LTBP) interact with the extracellular matrix and play an important role in the TGF-P activation process (for review see (Annes et al., 2003)). For BMPs a process identical to latency as observed for TGF-Ps is not known, but the pro-part of the BMPs possibly enhances the otherwise poor solubility of BMPs under physiolocigal conditions and thus might provide for or enhance their long-range activity (Sengle et al., 2008, Sengle et al., 2011). Recent determination of the structure of the TGF-P1 pro-protein now provides for an insight in the regulatory mechanism of the pro-part at atomic level (Shi et al., 2011). The pro-part embraces the mature part of TGF-P like a straitjacket, a long N-terminal a-helix binds into the type I receptor-binding site (in BMPs and GDFs called wrist epitope) thereby blocking receptor access to this epitope. A proline-rich loop termed latency lasso and a second a-helix encompass the fingertips and the back of the second finger of the mature part of TGF-P hence also blocking the type II receptor epitope. The pro-domain monomers form a dimerization site in the C-terminal region called bowtie, which is located above the butterfly-shaped dimeric TGF-P mature part. Two intermolecular disulfide bonds additionally stabilize the dimerization between the pro-domain subunits. Strikingly, the arrangement of the pro- and mature domain resembles the overall architecture found for the Noggin-BMP7 interaction (Groppe et al., 2002). Both receptor-binding epitopes are tightly blocked from receptor access and the binding of the modulator/pro-domain is strongly enhanced through avidity by forming a covalently linked dimer. The importance of the covalent dimer linkage becomes obvious in the rare bone disorder Camurati-Engelmann disease in which these cysteine residues in the TGF-P1 pro-part are mutated resulting in a disrupted dimerization and leading to increased ligand activation (Janssens et al., 2003, Walton et al., 2010).

Although the sequence homology (as well as differences in the length) between the prodomains of the various TGF-P members is certainly lower than between their mature parts alignments clearly show that all pro-domains will adopt a similar fold (Shi et al., 2011). A homology model for pro-GDF-5 build on the basis of pro-TGF-P1 structure instantly provides for possible explanations to why the effect of latency is quite different between TGF-Ps and members of the BMP subgroup. Particularly for GDF-5 (also true for GDF-6 and -7) many loops in the pro-domain are extended possibly creating further sites for proteolytic activation or degradation, secondly BMPs and GDFs lack the two cysteine residues present in the pro-domain being responsible for covalent linkage (see Fig. 9A). This suggests that the pro-domain association is much less stable for BMPs and GDFs (see mutations of cysteines in the Curati-Engelmann disease) and the release of the mature growth factor domain is facilitated without further need of processing. The four mutations in the GDF-5 pro-domain cluster in three different skeletal malformation phenotypes: M173V - BDC, S204R - BDC, R378Q/P436T (compound heterozygous) - Acromesomelic dysplasia, DuPan syndrome, R380Q - BDA2) indicating a loss-of-GDF-5 function in all cases (Everman et al., 2002, Schwabe et al., 2004, Douzgou et al., 2008, Ploger et al., 2008). On the basis of our own model methionine 173 is placed in close proximity to the first helix element blocking type I receptor binding, whereas serine 204 is placed in the so-called arm domain providing the structural scaffold for the straitjacket architecture. Both missense mutations likely lead to (local) unfolding and thus destabilize the pro-protein complex. This might subsequently lead to lower secretion efficiency and the observed loss-of-function phenotype. The mutation

Figure 9. Mutations in GDF-5 and its effect on structure or interactions. A) Homology model of pro-GDF-5 based on the structure of pro-TGF-P1 in ribbon representation (Shi et al., 2011). The mature part of GDF-5 (shown in blue and yellow) is embraced by the pro-part with the N-terminal part resembling a straitjacket (in red and orange). This element comprising of two helices block access to both type I and type II receptor binding epitopes. In contrast to the pro-part of TGF-Ps the pro-domains of BMPs and GDFs likely do not have intermolecular disulfides (the potential positions of Cys268 and Cys310 are shown) suggesting that the pro/mature part assembly of BMPs and GDFs might be less stable compared to TGF-Ps. Four missense mutations in the pro-part are found to be associated with skeletal malformation diseases: M173V, S204R, R378Q, and R380Q. The first two mutations (marked by green

Figure 9. Mutations in GDF-5 and its effect on structure or interactions. A) Homology model of pro-GDF-5 based on the structure of pro-TGF-P1 in ribbon representation (Shi et al., 2011). The mature part of GDF-5 (shown in blue and yellow) is embraced by the pro-part with the N-terminal part resembling a straitjacket (in red and orange). This element comprising of two helices block access to both type I and type II receptor binding epitopes. In contrast to the pro-part of TGF-Ps the pro-domains of BMPs and GDFs likely do not have intermolecular disulfides (the potential positions of Cys268 and Cys310 are shown) suggesting that the pro/mature part assembly of BMPs and GDFs might be less stable compared to TGF-Ps. Four missense mutations in the pro-part are found to be associated with skeletal malformation diseases: M173V, S204R, R378Q, and R380Q. The first two mutations (marked by green spheres) possibly cause misfolding of the pro-domain thereby weakening the pro-protein and leading to lower secretion efficiency. The latter two mutations are located in the furin protease site (marked as light-blue spheres) and were shown to lower or abrogate proteolytic processing of the pro-protein. B)

Homology model of the Noggin:GDF-5 complex (Schwaerzer et al., 2011) based on the crystal structure of the Noggin:BMP-7 complex (Groppe et al., 2002). Noggin, by a similar mechanism but different structural architecture, embraces GDF-5 thereby blocking receptor binding of either subtype through its clip and finger domains. Three missense mutations in GDF-5 associated with symphalangism were shown to have impaired GDF-5 - Noggin interaction: N445T/K, S475N, and E491K. All three mutations are in close proximity of the Noggin clip region suggesting that through loss of interaction with this element GDF-5 binding to Noggin is attenuated. C) Ribbon representation of the mature part of GDF-5 with the two monomeric subunits shown in blue and yellow. The architecture of a GDF-5 dimer resembles a left hand, the a-helix forming the palm, the two P-sheets depicting two fingers and the N-terminus marking the thumb. Consequentally, the receptor binding epitopes were named wrist (type I receptor), formed by the dorsal side of the fingers and the palm, and knuckle (type II receptor), formed by the ventral side of finger 1 and 2. The location of all known mutations associated with skeletal malformation diseases is depicted by spheres, with color-coding according to their belonging to either cystine knot mutations (red), pre-helix loop mutations (green) or mutations affecting Noggin-binding (magenta). D) As in C but rotated clockwise around the x-axis by 90°. E) Ribbon representation of the complex of GDF-5 (in blue and yellow) bound to the extracellular domain of BMPR-IB (grey). The overview clearly shows that affected residues in the pre-helix loop are in contact with receptor elements suggesting that these mutations alter type I receptor binding. F) Magnification of the interaction between residues in the pre-helix loop of GDF-5 and residues in the binding epitope of BMPR-IB. The complete pre-helix loop is tightly packed to residues in the threestranded P-sheet of BMPR-IB. GDF-5 Arg438 is involved in hydrogen bonds to His24 located in the P1P2-loop of BMPR-IB. The tight turn structures at the N- and C-terminal end of the pre-helix loop also indicate that the mutations involving the exchange of a proline (P436T) or introduction of a proline (L441P) will likely destroy the conformation of the pre-helix loop thereby affecting receptor binding even if these two residues do not form direct contacts with GDF-5.

R380Q targets the pro-domain cleavage site by destroying or attenuating proteolytic processing via furin proteases (Ploger et al., 2008). The now covalent linkage of pro- and mature part of GDF-5 R380Q very likely enhances the competition of the pro-domain with receptor binding and thus leads to loss of or attenuated GDF-5 activity (Ploger et al., 2008). The mechanism by which the double mutation R378Q/P436T causes the skeletal malformation is more complex. As the mutation is compound heterozygous, three GDF-5 variants are potentially produced in the patient. Statistically 50% of the GDF-5 protein would carry both exchanges as a heterodimer and the other 50% would consist of homodimers with either one of the two mutations. Heterozygous carriers of the individual missense mutations R378Q or P436T did not exhibit any skeletal phenotype thus preventing to point towards a particular mutation as disease-causing if found in a homozygous background. For the mutation R378Q it can be assumed that processing of the pro-protein is at least impaired and thus the portion of GDF-5 R378Q homodimer is likely to be inactive as found for R380Q (see Fig. 9) blank (Ploger et al., 2008). The missense mutation P436T is located in the mature part of GDF-5 in the so-called pre-helix loop of the GDF-5 type I receptor-binding epitope (Nickel et al., 2005). Mutation of the equivalent proline residue in BMP-2 strongly decreased binding of this BMP-2 variant to both type I receptors, BMPR-IA and BMPR-IB thus leading to a loss of BMP signaling (Kirsch et al., 2000).

Of the other eight known disease-related amino acid exchanges in the mature part of GDF-5, several mutations involve the exchange of a cysteine residue participating in the formation of the cystine knot, e.g. C400Y, C429R, C498S or introduce additional cysteine residues, e.g. R399C, R438C, which will interfere with proper formation of the cystine knot, thereby leading to a misfolded inactive protein. Several studies show that under conditions mimicking a homozygous background no secretion of the GDF-5 variant is observed (Everman et al., 2002, Dawson et al., 2006). However, mutations involving cysteines can also act dominant-negatively (see Fig. 9). Thomas et al. tested the effect of the GDF-5 mutation C400Y, which is found homozygous in chondrodysplasia Grebe type (Thomas et al., 1997). Upon transfection of only the mutated gene into COS-7 cells resembling a homozygous background no GDF-5 protein could be detected in the cell supernatant, however co-transfection of the genes for wildtype GDF-5 and the variant GDF-5 C400Y clearly attenuated GDF-5 protein levels in the supernatant. This effect was dose-dependent indicating that for heterozygous carriers through differential allelic expression a highly variable phenotype could possibly be observed (Thomas et al., 1997). Furthermore, this study also indicated that the mutation might act dominant negative onto other BMPs by selective heterodimerization. By co-transfection of the gene encoding for GDF-5 C400Y together with either BMP-2, BMP-3 or BMP-7, heterodimers could be isolated from the cell supernatant that will most likely be non-functional (Thomas et al., 1997).

3.6. GDF-5 activity is tightly regulated by the BMP antagonist Noggin

All other missense mutations in the GDF5 gene cluster in two regions of the GDF-5 structure (see Fig. 9C/D). Three missense mutations cluster in close proximity of finger 2 of GDF-5, N445T/K (Seemann et al., 2009), S475N (Akarsu et al., 1999, Schwaerzer et al., 2011) and E491K (Wang et al., 2006). The heterozygous mutations N445T and N445K in GDF-5 were identified in patients suffering from multiple synostosis syndrome (SYNS1) characterized by fusion of carpal bones and proximal symphalangism in fingers II to V (Seemann et al., 2009). Analysis of the recombinant GDF-5 variant in BMPR-IB transfected myoblastic C2C12 cells indicated that the mutation did not lead to a loss of GDF-5 function. In fact analyzing the expression of the osteogenic marker alkaline phosphatase in non-transfected C2C12 cells revealed even a gain of activity exemplified by a small but measureable ALP induction when stimulating with GDF-5 N445T but no induction of ALP expression when using wildtype GDF-5. As this activating mutation is located within the wrist (type I receptor binding) epitope of GDF-5 differences in binding to the BMP type I receptors were assumed. However, competition assays using soluble receptor ectodomains showed that binding of the GDF-5 variant N445T to BMPR-IA as well as BMPR-IB is unaltered (Seemann et al., 2009). Sequence comparison with other BMP factors indicated that one of the mutations found, the exchange of Asn445 to lysine, is native in BMP-9 and BMP-10. As the latter factors are insensitive to Noggin inhibition, Seemann et al. assumed that this mutation also renders GDF-5 insensitive to inhibition by Noggin. In vitro assays indeed confirmed that GDF-5 N445T is not antagonized by recombinant Noggin protein leading to an increase in GDF-5 signaling activity during early stages of limb and joint development where Noggin and GDF5 expression patterns overlap (Seemann et al., 2005, Seemann et al., 2009). Another mutation in GDF-5 leading to proximal symphalangism is

E491K discovered in two large Chinese families (Wang et al., 2006). The skeletal malformation phenotype resembles the one seen in aforementioned patients having either the mutation N445T/K (Seemann et al., 2009) or R438L (Seemann et al., 2005) in the GDF5 gene. Nothing is known about receptor or modulator protein binding of this particular GDF-5 variant, however in the GDF-5 structure Glu491 is in close proximity to Asn445. Moreover, the sidechain carboxamide group of Asn445 is forming a hydrogen bond to the backbone carbonyl of Glu491 possibly suggesting a similar disease-causing molecular mechanism through the loss of inhibition by Noggin as described above by Seemann et al. (2009). Modeling of a GDF-5:Noggin complex based on the structure of the BMP-7:Noggin interaction (Groppe et al., 2002) does however not indicate a direct interference of a GDF-5:Noggin interaction by exchanging Glu491 by lysine (see Fig. 9).

The mutation S475N is another mutation in the mature part of GDF-5, which causes multiple synostosis syndrome (SYNS1), a phenotypic description of these heterozygous missense mutations was first reported by Akarsu et al. (1999). The phenotype again suggests a gain-of-function in GDF-5 signaling. A detailed analysis of the signaling properties of this GDF-5 variant indeed revealed that GDF-5 S475N is significantly more potent in the chondrogenic differentiation in chicken micromass culture compared to wildtype GDF-5 (Schwaerzer et al., 2011). The mutation is located in the knuckle (type II receptor) epitope of GDF-5 (see Fig. 9C/D). Although no direct structural data is currently available for GDF-5 bound to type I and type II receptors, structure data available on ternary complexes of BMP-2 (Allendorph et al., 2006, Weber et al., 2007) indicated that this highly conserved serine residue is at the center of the BMP/GDF type II receptor interaction. Despite its location exchange of this residue in BMP-2 affected type II receptor binding only marginally (Weber et al., 2007) suggesting that other residues in the BMP-type II receptor interface are more important for the ligand-receptor interaction. However, in GDF-5 Ser475 seems more important for the binding of BMPR-II as indicated by a 7-fold decrease in the binding affinity upon mutation to asparagine, which seems surprising given the fact that this mutant shows an elevated activity compared to wildtype GDF-5 (Schwaerzer et al., 2011). As the BMP type II receptor epitope overlaps heavily with that of Noggin, also the change in binding to Noggin was determined showing that also Noggin binding affinity is similarly decreased by 4-fold. When the effect of Noggin inhibition on BMP factors was investigated by analyzing BMP-induced alkaline phosphatase expression or chondrogenic differentiation in chicken micromass culture in the presence of Noggin, GDF-5 S475N was clearly resistant to antagonizing effects by Noggin, whereas signals from wildtype GDF-5 could be efficiently blocked with Noggin (Schwaerzer et al., 2011). This possibly indicates that the loss in BMP type II receptor binding affinity seen for this variant is overcompensated by the deprivation of Noggin-mediated inhibition (Schwaerzer et al., 2011).

3.7. Type I receptor binding as well as receptor specificity is essential for correct GDF-5 function

A clear hotspot for disease-related mutations is found for the so-called pre-helix loop located in the wrist epitope of GDF-5 (Nickel et al., 2005). This loop is the key interaction element for BMP-type I receptor interaction (Kirsch et al., 2000, Keller et al., 2004, Kotzsch et al., 2008). For BMP-2 and GDF-5 this segment contains the so-called main binding determinant a highly conserved leucine residue, whose polar main chain atoms makes a pair of hydrogen bonds with a conserved glutamine residue present in the BMP type I receptors IA and IB. Mutation of either the leucine to a proline in BMP-2 or GDF-5 or the glutamine residue in BMPR-IA or BMPR-IB leads to a strongly reduced type I receptor affinity (Keller et al., 2004, Kotzsch et al., 2009). In the unbound state this pre-helix loop segment is also rather flexible allowing for geometrical adaptability to different receptor surface geometries. This observation together with the disordered and flexible ligand-binding epitope seen in the BMP type I receptors provides a mechanism for the pronounced ligand-receptor promiscuity seen in the BMP/GDF-subgroup of the TGF-P superfamily (Keller et al., 2004, Allendorph et al., 2007, Klages et al., 2008, Kotzsch et al., 2008, Saremba et al., 2008). Despite structural analyses showed that the pre-helix is flexible before receptor binding, the mutation L441P suggests that in the bound state a geometrically defined conformation is required for (high affinity) binding of BMP type I receptors (Kotzsch et al., 2009). Residue Leu441 is located at the C-terminal end of the pre-helix loop forming a sharp turn together with Ser439 and His440 (see Fig. 9E/F). The sidechain of Leu441 is oriented into the interior of GDF-5 making it implausible that its exchange to proline affects type I receptor binding through altering direct interactions. However, the different backbone torsion angle restraints of a non-proline compared to a proline residue suggest that the L441P mutation alters the conformation of the C-terminal end of the pre-helix loop and that hereby important non-covalent interactions between GDF-5 and its type I receptors are strongly impaired. Although earlier reports claim that the mutation L441P in GDF-5 affects binding to the BMP receptor IB (Faiyaz-Ul-Haque et al., 2002b, Seemann et al., 2005) our own data shows that binding to both BMP type I receptors is strongly attenuated (Kotzsch et al., 2009). A rather complex mutation discovered by Szczaluba et al. in patients suffering from DuPan syndrome shows shortening of all toes as well as all fingers but the thumb (Szczaluba et al., 2005). Here in the GDF-5 protein residue Leu437 is deleted and the adjacent residues Ser439 and His440 are mutated to threonine and leucine respectively (see Fig. 9). As these changes grossly alter the sequence as well as conformation of the pre-helix loop, it is not surprising that this GDF-5 compound variant shows no type I receptor binding at all (Kotzsch et al., 2009). Interestingly, although the mutation was found to be heterozygous in the carrier it has a dominant-negative effect (Szczaluba et al., 2005). Misfolding of the mutant protein and hence impaired secretion can be excluded as explanation, as the protein could be recombinantly produced and exhibits wildtype-like affinity to BMP type II receptors. One possible explanation for the quite strong skeletal phenotype might be that this GDF-5 variant is not only inactive but possibly still retains its Noggin-binding capability and therefore can act as a Noggin scavenger similar as to what was described for the BMP-2 variant L51P (Keller et al., 2004).

The probably most interesting mutation in GDF-5 is the exchange of Arg438 to leucine found in patients suffering from proximal symphalangism (Seemann et al., 2005). Based on a structural-function analysis to determine the GDF-5 type I receptor specificity this amino acid position - 438 if the complete pre-pro-protein is considered and position 57 if numbering starts with the mature part of GDF-5 - was shown before to be solely responsible for the BMPR-IB binding preference of GDF-5 (see Fig. 9E/F) (Nickel et al, 2005). The equivalent residue in BMP-2, which binds both BMP type I receptors, BMPR-IA and BMPR-IB, with equally high affinity is alanine. In contrast, in GDF-5 this position is occupied by a large positively charged arginine being also the largest difference in amino acid sequence within the central type I receptor-binding epitope. Upon exchange of Arg438 in GDF-5 to alanine, GDF-5 R438A bound both type I receptors with the same affinity and with binding characteristics indistinguishable from those of BMP-2 (Nickel et al., 2005). Recent structure analysis of GDF-5 bound to its type I receptor BMPR-IB revealed a molecular mechanism by which GDF-5 "discriminates" between both type I receptors (Kotzsch et al., 2009). A loop between the two N-terminal P-strands of the BMP type I receptors can adopt different conformations dependent on the amino acid sequence. As this loop is in contact to the "GDF-5 specificity determining" amino acid Arg438 BMP type I receptors can be selected through the presence or absence of a steric hindrance. BMPs with large bulky sidechains at this position such as GDF-5 of the pre-helix loop can only bind to BMPR-IB, whereas BMPs with small sidechains such as BMP-2 or BMP-4 can bind both BMP type I receptors equally well (Kotzsch et al., 2009).

Analysis of this BMP-2 like GDF-5 variant revealed that in a cell line (ATDC5) having pro-chondrogenic properties and not expressing the BMPR-IB receptor this variant now has the same signaling properties and efficiency as BMP-2 (Nickel et al., 2005). Thus under these conditions GDF-5 can signal via the BMPR-IA receptor and signaling efficiency is only decreased by the lower affinity of wildtype GDF-5 for BMPR-IA. Most interestingly, despite having the same receptor binding properties as BMP-2, GDF-5 R438A still does not induce ALP expression in the myoblastic cell line C2C12 (Klammert et al., 2011). As RT-PCR analysis did not reveal significant differences in BMP receptor expression between both cell lines, ATDC5 and C2C12, other mechanism must exist that determine whether GDF-5 can fully signal through a particular BMP type I receptor. This observation also indicates that GDF-5 by binding to BMPR-IA can activate signaling on some cell types whereas on other cell types it might compete with BMP-2 for BMPR-IA and act as an antagonist (Klammert et al., 2011). The mutation found in SYM1 affected humans, R438L, does not show a complete loss in BMP type I receptor specificity, the larger leucine sidechain in comparison to alanine leads to a 6 to 9-fold higher affinity to BMPR-IB compared to BMPR-IA (Seemann et al., 2005, Kotzsch et al., 2009). However, the result will likely be similar as above in that the mutation R438L renders GDF-5 into a protein that has BMP-2 like receptor binding properties. As BMP-2 is assumed to induce or at least regulate apoptosis in the interdigital mesenchyme (Yokouchi et al., 1996, Merino et al., 1999a), one would first expect increased apoptosis in patients carrying the mutation R438L in GDF-5 due to the presence of an additional BMP-2 like factor (Seemann et al., 2005). However, our latest observation that increased BMPR-IA binding by GDF-5 R438A might not induce full signaling in all cell types possibly indicates that here the gain-of-function mutation in GDF-5 surprisingly leads to a loss of BMP-2 signaling in certain areas of the developing joint by competing for the binding to the same receptor BMPR-IA thereby might impede BMP-2 induced apoptosis which finally results in joint fusion (Klammert et al., 2011).

4. Conclusion

When GDF-5 was discovered, due to its highly defined expression pattern during limb development, which precisely correlates with the location of all future joints throughout the limb, it was assumed immediately that this particular TGF-P factor takes the center stage in the development of all synovial joints. It thus came as a surprise when the GDF5 knockout mice despite being affected in joint and limb development still showed multiple joints being developed quite normally. Genetic and functional analyses of human skeletal malformation diseases such as brachydactyly or chondroplasia showed that not only a number of other genes can lead to loss of joints or limb deformations similar to those seen in the GDF5 null mice, but that also different mutations in GDF-5 can result in very distinct malformation phenotypes. Further studies revealed that often these different factors, many of them acting as morphogens themselves, such as Wnts and its (co-)receptors, members of the Sonic Hedgehog family or the FGFs, do not act independently but can be upstream or downstream of the TGF-P signaling cascade or even form positive or negative feedback loops with signaling components of the TGF-P superfamily. This complex regulatory network is further complicated by the fact that components of the TGF-P superfamily -ligands, receptors as well as antagonists - are known to function via highly promiscuous protein-protein interactions. Even if we restrict our focus onto the regulatory signaling network of GDF-5, its highly overlapping receptor binding specificities with other BMPs, such as BMP-2, BMP-6 or BMP-7, all of which are expressed in the direct neighborhood of the developing joint, make immediately clear that mutations altering binding of one particular ligand-receptor pair will ultimately affect the signaling output of other BMP members even when those are not affected by mutations themselves.

One mutation in GDF-5 - R438L - best exemplifies the dilemma. This mutation enables GDF-5 to now efficiently bind to a second BMP type I receptor, BMPR-IA. However this receptor is usually utilized by BMP-2 also present during joint development. As it is not known whether the GDF-5 variant with the altered type I receptor specificity delivers the same signal via this receptor as BMP-2 or whether it can signal at all through this BMP receptor in the present cellular context, developing a molecular disease mechanism explaining the mode of operation for this mutant seems impossible. In addition to this fuzzy BMP ligand-receptor network modulators like Noggin act like hub proteins interacting with multiple BMP ligands with a distinct BMP specificity profile. These interactions are again often linked to feedback loops leading to a precisely defined equilibrium of BMPs, BMP receptors and other modulators, which as a sum deliver a defined biological outcome. Classical morphogens such as the BMPs are considered to function via a concentration gradient, which is then interpreted by the different cells by responding to a particular morphogen threshold. However, the discrepancy of strong GDF5 expression in all future joint locations and the highly localized effect seen in GDF5 knockouts suggests that responsiveness to or the differentiation program run by GDF-5 is encoded along the digital ray by the various other morphogens in a temperospatial manner, thus allowing to run the differentiation program for joint formation by GDF-5 only at certain times at very defined places, whereas at other places or at earlier or later developmental stages as defined other factors will take over the GDF-5 function.

Author details

Tina V. Hellmann and Thomas D. Mueller

Dept. Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute of the University Wuerzburg, Wuerzburg, Germany

Joachim Nickel

Dept. Tissue Engineering and Regenerative Medicine, University Hospital Wuerzburg, Wuerzburg, Germany

Acknowledgement

We thank Markus Peer and Juliane E. Fiebig for helpful discussions and critically reading the manuscript.

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