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1.
Biointerphases ; 15(6): 061014, 2020 12 22.
Article in English | MEDLINE | ID: mdl-33353309

ABSTRACT

Precise characterization of a monolayer of two different biomolecules in a gradient pattern on a glass surface puts high demand on the method used. Some techniques can detect protein monolayers but not on a glass surface. Others can distinguish between different proteins but not identify a gradient pattern. Here, we used ToF-SIMS to validate the complete surface composition, checking all the necessary boxes. As these types of surfaces can dictate sensitive cell behaviors, the precision on a nanolevel is crucial, and to visualize and determine the molecular distribution become essential. The dual monolayer consisted of laminin 521 and one of three other biomolecules of different sizes, epidermal growth factor, growth differentiation factor 5, or bovine serum albumin, creating opposing gradient patterns. The resulting ToF-SIMS imaging and line scan data provided detailed information on the distribution of the adsorbed proteins.


Subject(s)
Epidermal Growth Factor/chemistry , Growth Differentiation Factor 5/chemistry , Serum Albumin, Bovine/chemistry , Spectrometry, Mass, Secondary Ion/methods , Adsorption , Animals , Cattle , Glass/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Surface Properties
2.
Theranostics ; 9(23): 6949-6961, 2019.
Article in English | MEDLINE | ID: mdl-31660079

ABSTRACT

Rationale: Articular cartilage injury is extremely common in congenital joint dysplasia patients. Genetic studies have identified Growth differentiation factor 5 (GDF5) as a shared gene in joint dysplasia and OA progression across different populations. However, few studies have employed GDF5 in biological regeneration for articular cartilage repair. Methods & Results: In the present study, we report identified genetic association between GDF5 loci and hip joint dysplasia with genome-wide association study (GWAS). GWAS and replication studies in separate populations achieved significant signals for GDF5 loci. GDF5 expression was dysregulated with allelic differences in hip cartilage of DDH and upregulated in the repaired cartilage in a rabbit cartilage defect model. GDF5 in vitro enhanced chondrogenesis and migration of bone marrow stem cells (BMSCs), GDF5 was tested in ectopic cartilage generation with BMSCs by GDF5 in nude mice in vivo. Genetically inspired, we further generated functional knee articular cartilage construct for cartilage repair by 3d-bioprinting a GDF5-conjugated BMSC-laden scaffold. GDF5-conjugated scaffold showed better cartilage repairing effects compared to control. Meanwhile, transplantation of the 3D-bioprinted GDF5-conjugated BMSC-laden scaffold in rabbit knees conferred long-term chondroprotection. Conclusions: In conclusion, we report identified genetic association between GDF5 and DDH with combined GWAS and replications, which further inspired us to generate a ready-to-implant GDF5-conjugated BMSC-laden scaffold with one-step 3d-bioprinting for cartilage repair.


Subject(s)
Bone Marrow Cells/metabolism , Cartilage, Articular/surgery , Growth Differentiation Factor 5/metabolism , Hip Dislocation/surgery , Hydrogels/chemistry , Stem Cells/metabolism , Tissue Scaffolds/chemistry , Animals , Bioprinting , Bone Marrow Transplantation , Cartilage, Articular/metabolism , Cell Movement , Chondrogenesis , Genome-Wide Association Study , Growth Differentiation Factor 5/chemistry , Hip Dislocation/genetics , Hip Dislocation/metabolism , Hip Dislocation/physiopathology , Humans , Mice , Mice, Nude , Printing, Three-Dimensional , Rabbits , Stem Cell Transplantation , Stem Cells/chemistry , Tissue Engineering
3.
Biophys Chem ; 240: 50-62, 2018 09.
Article in English | MEDLINE | ID: mdl-29890403

ABSTRACT

GDF-5 mediated signal transduction regulating chondrogenesis and skeletogenesis involves three different type-I receptors viz. Act-RI, BMPRIA and BMPRIB. BMPRIA and BMPRIB generally shows temporal and spatial co-expression but some spatially different expression pattern has also been observed. BMPRIA receptor is the key receptor implicated in BMP signalling during osteogenesis and is expressed in osteoblasts during the course of bone formation. However, BMPRIB appears to be primarily expressed in mesenchymal pre-cartilage condensations and also found in differentiated osteoblast and chondrocytes. The extracellular pH affects bone cell function and it is experimentally known that mineralization of bone is affected by shift of pH in cultured osteoblast. Here we report the effect of pH on dynamics of water present at the interface of GDF-5:BMPRIA and GDF-5:BMPRIB and binding interaction energy of these complexes. Water dynamics at different pH was analysed using residence time and hydrogen bond relaxation kinetics. pH influences the interaction energy between GDF-5 and BMPRIA and BMPRIB receptors indicating the electrostatic environment modulating the activity of two receptors. This pH dependence of interaction energy is further supported by similar behaviour of hydrogen bond existence of buried water molecules at the interface. In contrast to this the slow and fast exchanging water molecules do not show similar pH dependence of hydrogen bonding relaxation kinetics. Hence; we conclude that only buried water molecule at the interface influences the protein-protein interaction and the electrostatic environment of the extracellular fluid might decide the specificity of the two receptors.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/metabolism , Growth Differentiation Factor 5/metabolism , Water/chemistry , Amino Acid Sequence , Bone Morphogenetic Protein Receptors, Type I/chemistry , Bone Morphogenetic Protein Receptors, Type I/genetics , Growth Differentiation Factor 5/chemistry , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Sequence Alignment , Static Electricity
4.
Acta Biomater ; 72: 150-166, 2018 05.
Article in English | MEDLINE | ID: mdl-29550439

ABSTRACT

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor ß2 (TGF-ß2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-ß2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-ß2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-ß2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. STATEMENT OF SIGNIFICANCE: Regeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases.


Subject(s)
Adipose Tissue/metabolism , Fibroins/chemistry , Growth Differentiation Factor 5 , Ligaments , Mesenchymal Stem Cells/metabolism , Tendons , Tissue Scaffolds/chemistry , Transforming Growth Factor beta2 , Adipose Tissue/cytology , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/pharmacokinetics , Growth Differentiation Factor 5/pharmacology , Humans , Mesenchymal Stem Cells/cytology , Tissue Engineering , Transforming Growth Factor beta2/chemistry , Transforming Growth Factor beta2/pharmacokinetics , Transforming Growth Factor beta2/pharmacology
5.
J Biomed Mater Res B Appl Biomater ; 106(6): 2214-2224, 2018 08.
Article in English | MEDLINE | ID: mdl-29068568

ABSTRACT

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-ß with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2214-2224, 2018.


Subject(s)
Calcification, Physiologic/drug effects , Cartilage/metabolism , Fibrin , Growth Differentiation Factor 5 , Hydrogels , Mesenchymal Stem Cells/metabolism , Stem Cell Transplantation , Animals , Chondrogenesis/drug effects , Fibrin/chemistry , Fibrin/pharmacology , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/pharmacology , Heterografts , Humans , Hydrogels/chemistry , Hydrogels/pharmacology , Mice , Mice, SCID , Tissue Adhesives/chemistry , Tissue Adhesives/pharmacology
6.
Int J Mol Sci ; 18(8)2017 Aug 03.
Article in English | MEDLINE | ID: mdl-28771196

ABSTRACT

The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti) samples of disc and screw types using dexamethasone (DEX) and/or growth and differentiation factor-5 (GDF-5), as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8) assay, as well as the measurements of alkaline phosphatase (ALP) activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5-Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5-Ti and host bone, as evaluated by micro computed-tomography (micro CT) analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.


Subject(s)
Coated Materials, Biocompatible , Dexamethasone , Growth Differentiation Factor 5 , Heparin , Osteogenesis/drug effects , Titanium , beta-Cyclodextrins , Animals , Antigens, Differentiation/biosynthesis , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Dexamethasone/chemistry , Dexamethasone/pharmacology , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/pharmacology , Heparin/chemistry , Heparin/pharmacology , Humans , Mice , Rabbits , Titanium/chemistry , Titanium/pharmacology , beta-Cyclodextrins/chemistry , beta-Cyclodextrins/pharmacology
7.
Cell Rep ; 16(8): 2077-2086, 2016 08 23.
Article in English | MEDLINE | ID: mdl-27524626

ABSTRACT

The DAN family, including Gremlin-1 and Gremlin-2 (Grem1 and Grem2), represents a large family of secreted BMP (bone morphogenetic protein) antagonists. However, how DAN proteins specifically inhibit BMP signaling has remained elusive. Here, we report the structure of Grem2 bound to GDF5 at 2.9-Å resolution. The structure reveals two Grem2 dimers binding perpendicularly to each GDF5 monomer, resembling an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N terminus that undergoes significant transition upon complex formation, leading to simultaneous interaction with the type I and type II receptor motifs on GDF5. Binding studies show that DAN-family members can interact with BMP-type I receptor complexes, whereas Noggin outcompetes the type I receptor for ligand binding. Interestingly, Grem2-GDF5 forms a stable aggregate-like structure in vitro that is not clearly observed for other antagonists, including Noggin and Follistatin. These findings exemplify the structural and functional diversity across the various BMP antagonist families.


Subject(s)
Bone Morphogenetic Protein 2/chemistry , Growth Differentiation Factor 5/chemistry , Intercellular Signaling Peptides and Proteins/chemistry , Osteoblasts/metabolism , Proteins/chemistry , Amino Acid Motifs , Animals , Binding Sites , Binding, Competitive , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Crystallography, X-Ray , Cytokines , Embryo, Nonmammalian , Follistatin/chemistry , Follistatin/genetics , Follistatin/metabolism , Gene Expression , Growth Differentiation Factor 5/genetics , Growth Differentiation Factor 5/metabolism , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Osteoblasts/cytology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Proteins/genetics , Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenopus laevis
8.
J Nanosci Nanotechnol ; 16(1): 280-6, 2016 Jan.
Article in English | MEDLINE | ID: mdl-27398455

ABSTRACT

Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation.


Subject(s)
Cell Differentiation/drug effects , Coated Materials, Biocompatible , Growth Differentiation Factor 5 , Nanoparticles/chemistry , Osteogenesis/drug effects , Zirconium , Animals , Cell Line , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/pharmacology , Mice , Zirconium/chemistry , Zirconium/pharmacology
9.
Biochemistry ; 54(48): 7079-88, 2015 Dec 08.
Article in English | MEDLINE | ID: mdl-26562759

ABSTRACT

Protein-protein interactions are recognized as a fundamental phenomenon that is intimately associated with biological functions and thus are ideal targets for developing modulators for regulating biological functions. A challenge is to identify a site that is situated away from but functionally connected to the protein-protein interface. We employed bone morphogenetic proteins (BMPs) and their receptors as a model system to develop a strategy for identifying such a network of communication. Accordingly, using computational analyses with the COREX/BEST algorithm, we uncovered an overall pattern connecting various regions of BMPR-1B ectodomain, including the four conserved residues in the protein-protein interface. In preparation for testing the long-range effects of mutations of distal residues for future studies, we examined the extent of measurable perturbation of the four conserved residues by determination of the conformation and relative affinities of these BMPR-1B mutants for ligands BMP-2, -6, and -7 and GDF-5. Results suggest no significant structural changes in the receptor but do suggest that the four residues play different roles in defining ligand affinity and both intra- and intermolecular interactions play a role in defining ligand affinity. Thus, these results established two primary but necessary goals: (1) the baseline knowledge of perturbation of conserved interfacial residues for future reference and (2) the ability of the computational approach to identify the distal residues connecting to the interfacial residues. The data presented here provide the foundation for future experiments to identify the effects of distal residues that affect the specificity and affinity of BMP recognition. Protein-protein interactions are integral reactions in essentially all biological activities such as gene regulation and age-related development. Often, diseases are consequences of the alteration of these intermacromolecular interactions, which are thus recognized as a legitimate target for developing modulators for regulating biological functions. One approach is to design ligands that bind to the protein-protein interface. Another is to identify an allosteric site, an advantage of which is bypassing the potential challenge in competing for high-affinity interfacial interactions or a specific interface in a superassembly of multiple macromolecules. However, a challenge of this approach is identifying a site that is situated away from but functionally connected to the protein-protein interface.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/chemistry , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Proteins/metabolism , Growth Differentiation Factor 5/metabolism , Protein Interaction Maps , Alkaline Phosphatase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Proteins/chemistry , Cell Line , Conserved Sequence , Growth Differentiation Factor 5/chemistry , Humans , Mice , Models, Molecular , Protein Conformation , Protein Interaction Domains and Motifs , Protein Stability
10.
FEBS J ; 281(21): 4866-77, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25174448

ABSTRACT

The structure and function(s) of the very large proregions of the transforming growth factor-ß structure family are known in only a few cases. The proregion of growth and differentiation factor (GDF)5 comprises 354 residues. GDF5 therefore belongs to the group of those growth factors with the largest proregions. Here, we report a biophysical analysis of the proform (proGDF5) and the separate proregion. In the absence of the mature part, the proregion folds reversibly to form a monomeric polypeptide that is stabilized by an intramolecular disulfide bond. In the context of the mature part, i.e. in proGDF5, the proregion shows increased thermodynamic stability and contains a higher proportion of secondary structural elements than in its isolated form. A subdomain within the proregion represents a well-folded structure as monitored via biophysical analysis and NMR spectroscopy. Furthermore, two point mutations that are associated with skeletal malformations lead to reduced thermodynamic stability, which is interpreted on the basis of a homology model with the structure of the related latency-associated peptide, representing the proregion of transforming growth factor-ß1.


Subject(s)
Growth Differentiation Factor 5/chemistry , Amino Acid Substitution , Brachydactyly/genetics , Circular Dichroism , Cystine/analysis , Growth Differentiation Factor 5/drug effects , Growth Differentiation Factor 5/physiology , Hot Temperature , Humans , Models, Molecular , Mutation, Missense , Nuclear Magnetic Resonance, Biomolecular , Point Mutation , Protein Conformation , Protein Denaturation , Protein Folding , Protein Precursors/chemistry , Protein Processing, Post-Translational , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Spectrometry, Fluorescence , Structure-Activity Relationship
11.
Tissue Eng Part A ; 19(9-10): 1100-12, 2013 May.
Article in English | MEDLINE | ID: mdl-23157454

ABSTRACT

Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-derived factor 1α [SDF-1α], platelet-derived growth factor BB [PDGF-BB], insulin-like growth factor 1 [IGF-1], basic fibroblast growth factor [bFGF], and growth/differentiation factor 5 [GDF-5]) on the recruitment, proliferation, collagen synthesis, and genomic stability of equine tenocytes within an anisotropic collagen-GAG scaffold for tendon regeneration applications. Critically, we found that single factors led to a dose-dependent trade-off between driving tenocyte proliferation (PDGF-BB, IGF-1) versus maintenance of a tenocyte phenotype (GDF-5, bFGF). We identified supplementation schemes using factor pairs (IGF-1, GDF-5) to rescue the tenocyte phenotype and gene expression profiles while simultaneously driving proliferation. These results suggest coincident application of multi-biomolecule cocktails has a significant value in regenerative medicine applications where control of cell proliferation and phenotype are required. Finally, we demonstrated an immobilization strategy that allows efficient sequestration of bioactive levels of these factors within the scaffold network. We showed sequestration can lead to a greater sustained bioactivity than soluble supplementation, making this approach particularly amenable to in vivo translation where diffusive loss is a concern and continuous biomolecule supplementation is not feasible.


Subject(s)
Collagen/chemistry , Tendons/cytology , Tissue Scaffolds/chemistry , Animals , Biomechanical Phenomena , Cell Proliferation/drug effects , Cells, Cultured , Chemokine CXCL12/chemistry , Chemokine CXCL12/pharmacology , Fibroblast Growth Factor 2/chemistry , Fibroblast Growth Factor 2/pharmacology , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/pharmacology , Horses , Insulin-Like Growth Factor I/chemistry , Insulin-Like Growth Factor I/pharmacology , Platelet-Derived Growth Factor/chemistry , Platelet-Derived Growth Factor/pharmacology , Tissue Engineering/methods
12.
Biochim Biophys Acta ; 1824(12): 1374-81, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22824487

ABSTRACT

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-ß-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pK(a) analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin-BMP interactions.


Subject(s)
Bone Morphogenetic Proteins/chemistry , Heparin/metabolism , Amino Acid Sequence , Binding Sites , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 7/chemistry , Growth Differentiation Factor 5/chemistry , Molecular Docking Simulation , Molecular Sequence Data , Protein Structure, Secondary
13.
J Bone Miner Res ; 27(2): 429-42, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21976273

ABSTRACT

Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and non-Smad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs.


Subject(s)
Epitopes/genetics , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/genetics , Mutation/genetics , Synostosis/genetics , Amino Acid Sequence , Animals , Bone Morphogenetic Protein Receptors/metabolism , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Chondrogenesis/drug effects , Enzyme Activation/drug effects , Humans , Immobilized Proteins/pharmacology , Mice , Molecular Sequence Data , Mutant Proteins/metabolism , Myoblasts/drug effects , Myoblasts/metabolism , Myoblasts/pathology , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/pathology , Protein Binding/drug effects , Signal Transduction/drug effects , Smad Proteins/metabolism , Syndrome , Synostosis/enzymology , Synostosis/pathology , p38 Mitogen-Activated Protein Kinases/metabolism
14.
Article in English | MEDLINE | ID: mdl-21543859

ABSTRACT

The binary ligand-receptor complex of human growth and differentiation factor 5 (GDF5) bound to its type I receptor BMP receptor IA (BRIA) was prepared and crystallized. By utilizing the GDF5 variant R57A, which exhibits a high affinity in the subnanomolar range for BRIA, the binary complex of GDF5R57A bound to the extracellular domain of BRIA could be produced and purified. Crystals of this complex belonged to a monoclinic space group: either I2, with unit-cell parameters a = 63.81, b = 62.85, c = 124.99 Å, ß = 95.9°, or C2, with unit-cell parameters a = 132.17, b = 62.78, c = 63.53 Å, ß = 112.8°.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/chemistry , Growth Differentiation Factor 5/chemistry , Bone Morphogenetic Protein Receptors, Type I/isolation & purification , Crystallization , Crystallography, X-Ray , Growth Differentiation Factor 5/genetics , Growth Differentiation Factor 5/isolation & purification , Humans , Ligands , Mutation , Protein Binding
15.
Biomaterials ; 31(14): 3878-84, 2010 May.
Article in English | MEDLINE | ID: mdl-20170953

ABSTRACT

The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated beta-TCP carrier was compared to carriers coated with similar amounts (10 microg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.


Subject(s)
Calcium Phosphates/chemistry , Choristoma/pathology , Growth Differentiation Factor 5/genetics , Growth Differentiation Factor 5/pharmacology , Osteogenesis/drug effects , Point Mutation/genetics , Tissue Scaffolds/chemistry , Alkaline Phosphatase/metabolism , Animals , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Bone and Bones/enzymology , Bone and Bones/pathology , Enzyme Activation/drug effects , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/metabolism , Humans , Mice , Mice, SCID , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , X-Ray Microtomography
16.
BMC Biol ; 7: 59, 2009 Sep 07.
Article in English | MEDLINE | ID: mdl-19735544

ABSTRACT

BACKGROUND: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.


Subject(s)
Bone Morphogenetic Protein Receptors/chemistry , Bone Morphogenetic Protein Receptors/metabolism , Bone Morphogenetic Proteins/metabolism , Growth Differentiation Factor 5/metabolism , Protein Interaction Domains and Motifs/physiology , Activin Receptors/chemistry , Activin Receptors/genetics , Activin Receptors/isolation & purification , Activin Receptors/metabolism , Activins/chemistry , Activins/genetics , Activins/isolation & purification , Activins/metabolism , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Binding Sites , Biosensing Techniques , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Protein Receptors/isolation & purification , Bone Morphogenetic Proteins/chemistry , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/isolation & purification , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cholic Acids/chemistry , Detergents/chemistry , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/genetics , Growth Differentiation Factor 5/isolation & purification , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Kinetics , Ligands , Models, Biological , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Isoforms , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Transfection
17.
Article in English | MEDLINE | ID: mdl-19652338

ABSTRACT

The ligand-receptor complex of GDF5 bound to its type I and type II receptors BRIB and ActRIIB was produced and crystallized. Crystals of the GDF5-BRIB complex could only be obtained if a ternary complex comprising GDF5, BRIB and the extracellular domain of the type II receptor ActRIIB was used in crystallization; however, the type II receptor ActRIIB was lost during crystallization. Crystals of this complex belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 76.46, c = 82.78 A. Small changes in the crystallization condition resulted in crystals with a different morphology. These crystals consisted of the full ternary complex GDF5-BRIB-ActRIIB, but only diffracted to low resolution.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/chemistry , Growth Differentiation Factor 5/chemistry , Animals , Bone Morphogenetic Protein Receptors, Type I/isolation & purification , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Growth Differentiation Factor 5/isolation & purification , Humans , Ligands , Mice , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
18.
Anal Biochem ; 390(2): 103-8, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19393216

ABSTRACT

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor beta (TGF-beta) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP-HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-beta superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.


Subject(s)
Disulfides/analysis , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Alanine/chemistry , Alanine/genetics , Amino Acid Sequence , Amino Acid Substitution , Chromatography, High Pressure Liquid , Cysteine/chemistry , Cysteine/genetics , Escherichia coli/genetics , Humans , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Conformation , Protein Multimerization
19.
EMBO J ; 28(7): 937-47, 2009 Apr 08.
Article in English | MEDLINE | ID: mdl-19229295

ABSTRACT

Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-beta superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/metabolism , Growth Differentiation Factor 5/chemistry , Binding Sites , Bone Morphogenetic Protein Receptors, Type I/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Growth Differentiation Factor 5/metabolism , Humans , Models, Molecular , Mutation , Protein Conformation , Sensitivity and Specificity
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