Decorin interacts with connective tissue growth factor (CTGF)/CCN2 by LRR12 inhibiting its biological activity

Fibrotic disorders are the end point of many chronic diseases in different tissues, where an accumulation of the extracellular matrix occurs, mainly because of the action of the connective tissue growth factor (CTGF/CCN2). Little is known about how this growth factor activity is regulated. We found that decorin null myoblasts are more sensitive to CTGF than wild type myoblasts, as evaluated by the accumulation of fibronectin or collagen III. Decorin added exogenously negatively regulated CTGF pro-fibrotic activity and the induction of actin stress fibers. Using co-immunoprecipitation and in vitro interaction assays, decorin and CTGF were shown to interact in a saturable manner with a K(d) of 4.4 nM. This interaction requires the core protein of decorin. Experiments using the deletion mutant decorin indicated that the leucine-rich repeats (LRR) 10-12 are important for the interaction with CTGF and the negative regulation of the cytokine activity, moreover, a peptide derived from the LRR12 was able to inhibit CTGF-decorin complex formation and CTGF activity. Finally, we showed that CTGF specifically induced the synthesis of decorin, suggesting a mechanism of autoregulation. These results suggest that decorin interacts with CTGF and regulates its biological activity.     

The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles

Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.     

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.     

Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans

The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

The decorin sequence SYIRIADTNIT binds collagen type I

Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro. These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins.     

The carboxy-terminal pleckstrin homology domain of ROCK interacts with filamin-A

The small GTPase Rho and its effector ROCK/Rho-kinase regulate actin cytoskeletal reorganization through phosphorylation of the regulatory light chain of myosin II. We previously reported that ROCK co-purified with the actin-binding protein filamin-A from HeLa cells. Here, we show that the pleckstrin homology (PH) domain of ROCK, but not the kinase or coiled-coil domain, interacts with filamin-A. We also determined that the PH domain of ROCK binds to the carboxy-terminal region of filamin-A containing the last 24th repeat. ROCK co-localized with filamin-A at the protrusive cell membranes of HeLa cells.     

Leucine-rich repeat (LRR) proteins: integrators of pattern recognition and signaling in immunity

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2-45 leucine-rich repeats, with each repeat about 20-30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.     

Leucine-rich repeat (LRR) proteins: Integrators of pattern recognition and signaling in immunity

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2–45 leucine-rich repeats, with each repeat about 20–30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.     

Filamin-A as a marker and target for DNA damage based cancer therapy

Filamin-A, also called actin binding protein 280 (ABP-280), cross-links the actin filaments into dynamic orthogonal network to serve as scaffolds in multiple signaling pathways. It has been reported that filamin-A interacts with DNA damage response proteins BRCA1 and BRCA2. Defects of filamin-A impair the repair of DNA double strand breaks (DSBs), resulting in sensitization of cells to ionizing radiation. In this study, we sought to test the hypothesis that filamin-A can be used as a target for cancer chemotherapy and as a biomarker to predict cancer response to therapeutic DNA damage. We found that reduction of filamin-A sensitizes cancer cells to chemotherapy reagents bleomycin and cisplatin, delays the repair of not only DSBs but also single strand breaks (SSBs) and interstrand crosslinks (ICLs), and increases chromosome breaks after the drug treatment. By treating a panel of human melanoma cell lines with variable filamin-A expression, we observed a correlation between expression level of filamin-A protein and drug IC(50). We further inhibited the expression of filamin-A in melanoma cells, and found that this confers an increased sensitivity to bleomycin and cisplatin treatment in a mouse xenograft tumor model. These results suggest that filamin-A plays a role in repair of a variety of DNA damage, that lack of filamin-A is a prognostic marker for a better outcome after DNA damage based treatment, and filamin-A can be inhibited to sensitize filamin-A positive cancer cells to therapeutic DNA damage. Thus filamin-A can be used as a biomarker and a target for DNA damage based cancer therapy.     

Fibromodulin binds collagen type I via Glu-353 and Lys-355 in leucine-rich repeat 11

Fibromodulin belongs to the small leucine-rich repeat proteoglycan family, interacts with collagen type I, and controls collagen fibrillogenesis and assembly. Here, we show that a major fibromodulin-binding site for collagen type I is located in leucine-rich repeat 11 in the C terminus of the leucine-rich repeat domain. We identified Glu-353 and Lys-355 in repeat 11 as essential for binding, and the synthetic peptide RLDGNEIKR, including Glu-353 and Lys-355, inhibits the binding of fibromodulin to collagen in vitro. Fibromodulin and lumican compete for the same binding region on collagen, and fibromodulin can inhibit the binding of lumican to collagen type I. However, the peptide RLDGNEIKR does not inhibit the binding of lumican to collagen, suggesting separate but closely situated fibromodulin- and lumican-binding sites in collagen. The collagen-binding Glu-353 and Lys-355 residues in fibromodulin are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat, which resembles the location of interacting residues in other leucine-rich repeat proteins, e.g. decorin.     

An anti-oncogenic role for decorin. Down-regulation of ErbB2 leads to growth suppression and cytodifferentiation of mammary carcinoma cells

The leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor and triggers a signaling pathway that leads to growth suppression. We find that decorin causes a functional inactivation of the oncogenic ErbB2 protein in breast carcinoma cells. Upon de novo expression of decorin, the ErbB2 protein is reduced by approximately 40%, whereas its degree of tyrosyl phosphorylation is almost completely abrogated. Both co-culture experiments or experiments with recombinant decorin demonstrate an initial induction of ErbB2 tyrosine kinase, followed by a profound and long-lasting down-regulation of its activity. This leads to growth inhibition and cytodifferentiation of mammary tumor cells and a concurrent suppression of their tumorigenic potential in vivo. These decorin-mediated effects appear to involve the activation of ErbB4, which in turn would block the phosphorylation of heterodimers containing either ErbB2 or ErbB3. These results provide an explanation for the heightened decorin levels around invasive carcinomas and suggest that decorin may function as a natural antagonist of neoplastic cells enriched in ErbB2.     

Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation

Plant nucleotide binding and leucine-rich repeat (NB-LRR) proteins contain a region of homology known as the ARC domain located between the NB and LRR domains. Structural modeling suggests that the ARC region can be subdivided into ARC1 and ARC2 domains. We have used the potato (Solanum tuberosum) Rx protein, which confers resistance to Potato virus X (PVX), to investigate the function of the ARC region. We demonstrate that the ARC1 domain is required for binding of the Rx N terminus to the LRR domain. Domain-swap experiments with Rx and a homologous disease resistance gene, Gpa2, showed that PVX recognition localized to the C-terminal half of the LRR domain. However, inappropriate pairings of LRR and ARC2 domains resulted in autoactive molecules. Thus, the ARC2 domain is required to condition an autoinhibited state in the absence of elicitor as well as for the subsequent elicitor-induced activation. Our data suggest that the ARC region, through its interaction with the LRR, translates elicitor-induced modulations of the C terminus into a signal initiation event. Furthermore, we demonstrate that physical disruption of the LRR-ARC interaction is not required for signal initiation. We propose instead that this activity can lead to multiple rounds of elicitor recognition, providing a means of signal amplification.     

cDNA clone to chick corneal chondroitin/dermatan sulfate proteoglycan reveals identity to decorin.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.     

Structural diversity of the hagfish variable lymphocyte receptors

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.     

Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.     

Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.     

Decorin binds to a narrow region of the epidermal growth factor (EGF) receptor,partially overlapping but distinct from the EGF-binding epitope

Decorin, a small leucine-rich proteoglycan, is a key regulator of tumor growth by acting as an antagonist of the epidermal growth factor receptor (EGFR) tyrosine kinase. To search for cell surface receptors interacting with decorin, we generated a decorin/alkaline phosphatase chimeric protein and used it to screen a cDNA library by expression cloning. We identified two strongly reactive clones that encoded either the full-length EGFR or its ectodomain. A physiologically relevant interaction between decorin and EGFR was confirmed in the yeast two-hybrid system and further validated by experiments using EGF/EGFR interaction and transient cell transfection assays. Using a panel of deletion mutants, decorin binding was mapped to a narrow region of the EGFR within its ligand-binding L2 domain. Moreover, the central leucine-rich repeat 6 of decorin was required for interaction with the EGFR. Site-directed mutagenesis of the EGFR L2 domain showed that a cluster of residues, His(394)-Ile(402), was essential for both decorin and EGF binding. In contrast, K465, previously shown to be cross-linked to epidermal growth factor (EGF), was required for EGF but not for decorin binding. Thus, decorin binds to a discrete region of the EGFR, partially overlapping with but distinct from the EGF-binding domain. These findings could lead to the generation of protein mimetics capable of suppressing EGFR function.     

Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the Ascidian Ciona intestinalis

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.