Sequence features,structure, ligand interaction,and diseases in small leucine rich repeat proteoglycans

Small leucine rich repeat proteoglycans (SLRPs) are a group of active components of the extracellular matrix in all tissues. SLRPs bind to collagens and regulate collagen fibril growth and fibril organization. SLRPs also interact with various cytokines and extracellular compounds, which lead to various biological functions such cell adhesion and signaling, proliferation, and differentiation. Mutations in SLRP genes are associated with human diseases. Now crystal structures of five SLRPs are available. We describe some features of amino acid sequence and structures of SLRPs. We also review ligand interactions and then discuss the interaction surfaces. Furthermore, we map mutations associated with human diseases and discuss possible effects on structures by the mutations.     

Small leucine-rich proteoglycans in the aging skeleton

Small Leucine-Rich Proteoglyans (SLRPs) are major skeletal extracellular matrix (ECM) components that comprise a family of 13 members containing repeats of a leucine-rich motif. To examine SLRP function, we generated mice deficient in one or more member and analyzed them at the tissue, cell and molecular levels. This review outlines the novel research findings uncovered using these new animal models.     

Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction

The small leucine-rich proteoglycan (SLRP) family has significantly expanded in the past decade to now encompass five discrete classes, grouped by common structural and functional properties. Some of these gene products are not classical proteoglycans, whereas others have new and unique features. In addition to being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple signaling cascades. They affect intracellular phosphorylation, a major conduit of information for cellular responses, and modulate distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor beta superfamily members, receptor tyrosine kinases such as ErbB family members and the insulin-like growth factor I receptor, and Toll-like receptors. The wealth of mechanistic insights into the molecular and cellular functions of SLRPs has revealed both the sophistication of this family of regulatory proteins and the challenges that remain in uncovering the totality of their functions. This review is focused on novel biological functions of SLRPs with special emphasis on their protein cores, newly described genetic diseases, and signaling events in which SLRPs play key functions.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

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.     

The matricellular functions of small leucine-rich proteoglycans (SLRPs)

The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFα), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-β, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.     

Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis,osteoarthritis, Ehlers-Danlos syndrome,muscular dystrophy,and corneal diseases

Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.     

Inhibition of hydroxyapatite crystal growth by bone proteoglycans and proteoglycan components

The small leucine-rich proteoglycans (SLRPs) interact with hydroxyapatite (HAP) and have been demonstrated to be important modulators of mineralisation. In the present study we have examined the effect of bone SLRPs, purified bone glycosaminoglycan (GAG) chains and core proteins as well as commercial chondroitin 4-sulphate, chondroitin 6-sulphate and desulphated chondroitin on HAP crystal growth. Seeded HAP growth experiments revealed that addition of bone GAG chains resulted in almost complete inhibition of crystal growth (93%), with addition of core proteins and intact PGs resulting in 55 and 37% inhibition, respectively. In contrast, commercial chondroitin 4-sulphate was significantly less inhibitory compared with the bone SLRPs and components, yielding only a 6% reduction in HAP-induced crystal growth at the same concentration. Significantly, chondroitin 6-sulphate was found to be noninhibitory, whilst desulphated chondroitin was inhibitory to seeded HAP growth. The data indicate that direct adsorption of SLRPs to growth sites and their ability to bind calcium are significant determinants in the inhibitory process. In addition, PG/GAG chemistry and the conformation of the macromolecules in solution have also been shown to be important. This work provides new information regarding the role of bone SLRPs and their components in the regulation of the mineralisation process.     

Proteoglycans of the extracellular matrix and growth control.

Regulated cell growth results from the biological balance between soluble growth-regulating factors, their receptors and the elicited signal cascade on the one hand side and from extracellular macromolecular components and their interplay with membrane receptors on the other side. Proteoglycans have recently been recognized not only to play a part in providing shape and biomechanical strength of organs and tissues, but also to exhibit direct and indirect cell signalling properties. In this review, we discuss the direct growth-regulating role of proteoglycans with special emphasis on the lectican family and on the family of small proteoglycans with leucine-rich repeats (SLRPs). Indirect actions of proteoglycans by modulation of growth factor activities and growth factor distribution are exemplified by discussing the TGF-beta-binding properties of SLRPs and the interactions of core proteins of matrix proteoglycans with other growth factors. It is emphasized that the modulatory role of proteoglycans on cell proliferation cannot be separated from their participation in tissue organization in general, thereby explaining the diverse and sometimes contradictory reports on the effects of proteoglycans on cell proliferation and differentiation.     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G₁₇₇/V₁₇₈) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Keratocan-deficient mice display alterations in corneal structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.     

Osteoadherin accumulates in the predentin towards the mineralization front in the developing tooth

Background

Proteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated.

Methodology/Principal Findings

We investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization.

Conclusions

These studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.     

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, “Podocan-like (Podnl) protein” identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.     

Differential regulation of osteoadherin (OSAD) by TGF-beta1 and BMP-2

Osteoadherin (OSAD) is a member of the small leucine rich-repeat proteoglycan (SLRP) family. SLRPs are normally found in extracellular matrices, but OSAD is the only member restricted to mineralized tissues. We investigated the promoter region of OSAD by in silico analysis and found that the proximal promoter region contains sites for Smad-3, Smad-4, and AP-1. All are effectors of TGF-beta family signalling. We tested sensitivity of the promoter to the two TGF-beta family members TGF-beta1 and BMP-2. We found TGF-beta1 to down regulate OSAD, while BMP-2 up regulates OSAD. As a consequence of how OSAD is regulated by TGF-beta1 and BMP-2 and its temporal expression pattern in osteoblasts and bone development, we can conclude OSAD as an early marker for terminally differentiated matrix producing osteoblasts.     

The Biology of Small Leucine-rich Proteoglycans in Bone Pathophysiology

The class of small leucine-rich proteoglycans (SLRPs) is a family of homologous proteoglycans harboring relatively small (36–42 kDa) protein cores compared with the larger cartilage and mesenchymal proteoglycans. SLRPs have been localized to most skeletal regions, with specific roles designated during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition. This is mediated by key signaling pathways regulating the osteogenic program, including the activities of TGF-β, bone morphogenetic protein, Wnt, and NF-κB, which influence both the number of available osteogenic precursors and their subsequent development, differentiation, and function. On the other hand, SLRP depletion is correlated with degenerative diseases such as osteoporosis and ectopic bone formation. This minireview will focus on the SLRP roles in bone physiology and pathology.     

Comparative analysis of human matrix metalloproteinases: Emerging therapeutic targets in diseases

The identification of specific target proteins for any diseased condition involves extensive characterization of the potentially involved proteins. Members of a protein family demonstrating comparable features may show certain unusual features when implicated in a pathological condition. Advancements in the field of computational biology and the use of various bioinformatics tools for analysis can aid researchers to comprehend their system of work in primary stages of research. This initial screening can help to reduce time and cost of testing and experimentation in laboratory. Human matrix metalloproteinase (MMP) family of endopeptidases is one such family of 23 members responsible for the remodeling of extracellular matrix (ECM) by degradation of the ECM proteins. Though their role has been implicated in various pathological conditions such as arthritis, atherosclerosis, cancer, liver fibrosis, cardio-vascular and neurodegenerative disorders, little is known about the specific involvement of members of the large MMP family in diseases. A comparative in silico characterization of the MMP protein family has been carried out to analyze their physico-chemical, secondary structural and functional properties. Based on the observed patterns of occurrence of atypical features, we hypothesize that cysteine rich and highly thermostable MMPs might be key players in diseased conditions. Thus, a plausible grouping of disease responsive MMPs that might be considered as promising clinical targets may be done. This study can be used as a fundamental approach to characterize, analyze and screen large protein families for the identification of signature patterns.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.