A novel homozygous missense variant in MATN3 causes spondylo-epimetaphyseal dysplasia Matrilin 3 type in a consanguineous family.

Spondylo-epimetaphyseal dysplasia Matrilin 3 type (SEMD) is a rare autosomal recessive skeletal dysplasia characterized by short stature, abnormalities in the vertebral bodies and long bones, especially the lower limbs. We enrolled a consanguineous family from Pakistan in which multiple siblings suffered from severe skeletal dysplasia. The six affected subjects ranged in heights from 100 to 136 cm (~-6 standard deviation). Lower limb abnormalities with variable varus and valgus deformities and joint dysplasia were predominant features of the clinical presentation. Whole exome sequencing (WES) followed by Sanger sequencing identified a missense variant, c.542G > A, p.(Arg181Gln) in MATN3 as the genetic cause of the disorder. The variant was homozygous in all affected individuals while the obligate carriers had normal heights with no skeletal symptoms, consistent with a recessive pattern of inheritance. Multiple sequence alignment revealed that MATN3 domain affected by the variant is highly conserved in orthologous proteins. The c.542G > A, p.(Arg181Gln) variant is only the fourth variant in MATN3 causing an autosomal recessive disorder and thus expands the genotypic spectrum.

XBP1 signalling is essential for alleviating mutant protein aggregation in ER-stress related skeletal disease.

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific 'disease signature' of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation.

Suppressing mesenchymal stem cell hypertrophy and endochondral ossification in 3D cartilage regeneration with nanofibrous poly(l-lactic acid) scaffold and matrilin-3.

Articular cartilage has a very limited ability to self-heal after injury or degeneration due to its low cellularity, poor proliferative activity, and avascular nature. Current clinical options are able to alleviate patient suffering, but cannot sufficiently regenerate the lost tissue. Biomimetic scaffolds that recapitulate the important features of the extracellular matrix (ECM) of cartilage are hypothesized to be advantageous in supporting cell growth, chondrogenic differentiation, and integration of regenerated cartilage with native cartilage, ultimately restoring the injured tissue to its normal function. It remains a challenge to support and maintain articular cartilage regenerated by bone marrow-derived mesenchymal stem cells (BMSCs), which are prone to hypertrophy and endochondral ossification after implantation in vivo. In the present work, a nanofibrous poly(l-lactic acid) (NF PLLA) scaffold developed by our group was utilized because of the desired highly porous structure, high interconnectivity, and collagen-like NF architecture to support rabbit BMSCs for articular cartilage regeneration. We further hypothesized that matrilin-3 (MATN3), a non-collagenous, cartilage-specific ECM protein, would enhance the microenvironment of the NF PLLA scaffold for cartilage regeneration and maintain the cartilage property. To test this hypothesis, we seeded BMSCs on the NF PLLA scaffold with or without MATN3. We found that MATN3 suppresses hypertrophy in this 3D culture system in vitro. Subcutaneous implantation of the chondrogenic cell/scaffold constructs in a nude mouse model showed that pretreatment with MATN3 was able to maintain chondrogenesis and prevent hypertrophy and endochondral ossification in vivo. These results demonstrate that the porous NF PLLA scaffold treated with MATN3 represents an advantageous 3D microenvironment for cartilage regeneration and phenotype maintenance, and is a promising strategy for articular cartilage repair. STATEMENT OF SIGNIFICANCE: Articular cartilage defects, caused by trauma, inflammation, or joint instability, may ultimately lead to debilitating pain and disability. Bone marrow-derived mesenchymal stem cells (BMSCs) are an attractive cell source for articular cartilage tissue engineering. However, chondrogenic induction of BMSCs is often accompanied by undesired hypertrophy, which can lead to calcification and ultimately damage the cartilage. Therefore, a therapy to prevent hypertrophy and endochondral ossification is of paramount importance to adequately regenerate articular cartilage. We hypothesized that MATN3 (a non-collagenous ECM protein expressed exclusively in cartilage) may improve regeneration of articular cartilage with BMSCs by maintaining chondrogenesis and preventing hypertrophic transition in an ECM mimicking nanofibrous scaffold. Our results showed that the administration of MATN3 to the cell/nanofibrous scaffold constructs favorably maintained chondrogenesis and prevented hypertrophy/endochondral ossification in the chondrogenic constructs in vitro and in vivo. The combination of nanofibrous PLLA scaffolds and MATN3 treatment provides a very promising strategy to generate chondrogenic grafts with phenotypic stability for articular cartilage repair.

Matrilins.

Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation. Due to their intrinsic adhesiveness and partially also low abundance, the study of matrilins is cumbersome. In this chapter, we describe methods for purification of matrilins from tissue, analysis of matrilins in tissue extracts, recombinant expression, and generation of matrilin-specific antibodies.

Characterization of recombinantly expressed matrilin VWA domains.

VWA domains are the predominant independent folding units within matrilins and mediate protein-protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.

Matrilin-3 inhibits chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist.

Increased chondrocyte hypertrophy is often associated with cartilage joint degeneration in human osteoarthritis patients. Matrilin-3 knock-out (Matn3 KO) mice exhibit these features. However, the underlying mechanism is unknown. In this study, we sought a molecular explanation for increased chondrocyte hypertrophy in the mice prone to cartilage degeneration. We analyzed the effects of Matn3 on chondrocyte hypertrophy and bone morphogenetic protein (Bmp) signaling by quantifying the hypertrophic marker collagen type X (Col X) gene expression and Smad1 activity in Matn3 KO mice in vivo and in Matn3-overexpressing chondrocytes in vitro. The effect of Matn3 and its specific domains on BMP activity were quantified by Col X promoter activity containing the Bmp-responsive element. Binding of MATN3 with BMP-2 was determined by immunoprecipitation, solid phase binding, and surface plasmon resonance assays. In Matn3 KO mice, Smad1 activity was increased more in growth plate chondrocytes than in wild-type mice. Conversely, Matn3 overexpression in hypertrophic chondrocytes led to inhibition of Bmp-2-stimulated, BMP-responsive element-dependent Col X expression and Smad1 activity. MATN3 bound BMP-2 in a dose-dependent manner. Multiple epidermal growth factor (EGF)-like domains clustered together by the coiled coil of Matn3 is required for Smad1 inhibition. Hence, as a novel BMP-2-binding protein and antagonist in the cartilage extracellular matrix, MATN3 may have the inherent ability to inhibit premature chondrocyte hypertrophy by suppressing BMP-2/Smad1 activity.

Matrilin-2 is proteolytically cleaved by ADAMTS-4 and ADAMTS-5.

Matrilin-2 is a widely distributed, oligomeric extracellular matrix protein that forms a filamentous network by binding to a variety of different extracellular matrix proteins. We found matrilin-2 proteolytic products in transfected cell lines in vitro and in mouse tissues in vivo. Two putative cleavage sites were identified in the unique domain of matrilin-2; the first site was located between D851 and L852 in the middle of the domain and the second, at the boundary with the coiled-coil domain at the C-terminus. Deletion of the entire unique domain eliminated the proteolysis of matrilin-2. While the first cleavage site was present in all matrilin-2 oligomers, the second cleavage site became apparent only in the matrilin-2 hetero-oligomers with matrilin-1 or matrilin-3. Analysis using a variety of extracellular protease inhibitors suggested that this proteolytic activity was derived from a member or several members  of the ADAMTS family. Recombinant human ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2), but not ADAMTS-1, cleaved recombinant matrilin-2, thereby yielding matrilin-2 proteolytic peptides at the predicted sizes. These results suggest that ADAMTS-4 and ADAMTS-5 may destabilize the filamentous network in the extracellular matrix by cleaving matrilin-2 in both homo-oligomers and hetero-oligomers.

Matrilin-1 is an inhibitor of neovascularization.

In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.

Tribody: robust self-assembled trimeric targeting ligands with high stability and significantly improved target-binding strength.

The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribodies), against epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target-binding strength compared with the corresponding monomers. Cellular-binding studies confirmed that the trimeric targeting ligands have superior binding strength toward their respective receptors. Significantly, the EGFR-binding tribody was considerably accumulated in the tumor of mice bearing xenografted EGFR-positive tumors, indicating its effective cancer-targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribodies can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.