ANTXR1 deficiency promotes fibroblast senescence: implications for GAPO syndrome as a progeroid disorder.

ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1's physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging.

The Fraser Complex Proteins (Frem1, Frem2, and Fras1) Can Form Anchoring Cords in the Absence of AMACO at the Dermal-Epidermal Junction of Mouse Skin.

AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal-epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.

Structure, evolution and expression of zebrafish cartilage oligomeric matrix protein (COMP, TSP5). CRISPR-Cas mutants show a dominant phenotype in myosepta.

COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Lack of evidence for a role of anthrax toxin receptors as surface receptors for collagen VI and for its cleaved-off C5 domain/endotrophin.

The microfibril-forming collagen VI is proteolytically cleaved and it was proposed that the released C-terminal Kunitz domain (C5) of the α3 chain is an adipokine important for tumor progression and fibrosis. Designated "endotrophin," C5 is a potent biomarker for fibroinflammatory diseases. However, the biochemical mechanisms behind endotrophin activity were not investigated. Earlier, anthrax toxin receptor 1 was found to bind C5, but this potential interaction was not further studied. Given the proposed physiological role of endotrophin, we aimed to determine how the signal is transmitted. Surprisingly, we could not detect any interaction between endotrophin and anthrax toxin receptor 1 or its close relative, anthrax toxin receptor 2. Moreover, we detect no binding of fully assembled collagen VI to either receptor. We also studied the collagen VI receptor NG2 (CSPG4) and confirmed that NG2 binds assembled collagen VI, but not cleaved C5/endotrophin. A cellular receptor for C5/endotrophin, therefore, still remains elusive.

Anchoring Cords: A Distinct Suprastructure in the Developing Skin.

AMACO (VWA2 protein) is a basement membrane-associated protein secreted by epithelial cells. It is strongly expressed when invagination or budding occurs during development. AMACO associates with the Fraser complex, which when mutated causes Fraser syndrome, characterized by subepidermal blistering, cryptophthalmos, and syndactyly. The core Fraser complex proteins FRAS1, FREM1, and FREM2 localize at the dermal‒epidermal junction and mediate adhesion to the underlying dermis during embryonic development. Earlier transmission electron microscopy studies of adult mouse skin showed clustered AMACO deposition below the lamina densa. In this study, we report a distinct cord-like suprastructure in the neonate dermis to which AMACO- and Fraser complex‒associated proteins contribute. We propose anchoring cords to designate the suprastructure. Anchoring cords have a diameter of 60 nm when immunolabeled, originate from the basement membrane, and extend several microns into the dermis. In normal skin, they are evident after immunogold electron microscopy and are strikingly appreciated in thicker sections. In recessive dystrophic epidermolysis bullosa skin, they are directly visible where collagen VII anchoring fibrils are ablated. Immunofluorescence and coimmunoprecipitation of skin extracts identify a direct interaction of FREM2 and AMACO.

LTBP1 promotes fibrillin incorporation into the extracellular matrix.

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFß growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFß-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Collagen type VI is the antigen recognized by the ER-TR7 antibody.

The monoclonal antibody ER-TR7 was used in a great number of studies for detecting reticular fibroblasts and the ECM of lymphoid and non-lymphoid organs even if the protein recognized by the ER-TR7 antibody was not known. We have now identified native collagen VI microfibrils as its tissue antigen.

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2.

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

Author Correction: Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis.

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4-/- mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4-/- mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4-/- mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4-/- mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

Spatio-temporal expression and distribution of collagen VI during zebrafish development.

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.

Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing.

The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFß)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.

C-terminal proteolysis of the collagen VI α3 chain by BMP-1 and proprotein convertase(s) releases endotrophin in fragments of different sizes.

The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies.

The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI-related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.

Identification of antibodies against extracellular matrix proteins in human osteoarthritis.

We investigated the presence of autoantibodies against the extracellular matrix proteins thrombospondin-4 (TSP-4), cartilage oligomeric matrix protein (COMP), C-type lectin domain family 3 member A (CLEC3A), collagen II, collagen VI, matrilin-3, and fibrillin-2 in the serum of osteoarthritis (OA) patients. We compared those results with the presence of such antibodies in rheumatoid arthritis (RA) patients and in healthy donors (HD). Our study examines whether antibodies against extracellular proteins can be used as potential biomarkers to support the clinical diagnosis of OA. 10 OA, 10 RA patients and 10 HD were enrolled in this explorative cross-sectional study. SDS-PAGE and immunoblot were used to investigate the presence of antibodies against extracellular matrix proteins. The serum of 5/10 OA patients but 0/10 HD exhibited TSP-4 IgG isotype antibodies (P = 0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (P = 0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (P = 0.005). We found strong evidence for the presence of IgG isotype autoantibodies against the cartilage extracellular matrix proteins TSP-4, COMP and CLEC3A in OA. The detection of IgG isotype autoantibodies against TSP-4, COMP and CLEC3A may support the clinical diagnosis of OA. OA with autoantibodies against cartilage extracellular matrix proteins defines a new OA subgroup suggesting that patients with high concentrations of autoantibodies may benefit from an immune suppressive therapy.

Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity.

Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.

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.