ABSTRACT
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.
Subject(s)
Alopecia , Anodontia , Cellular Senescence , Fibroblasts , Growth Disorders , Microfilament Proteins , Humans , Fibroblasts/metabolism , Cellular Senescence/genetics , Alopecia/metabolism , Alopecia/pathology , Alopecia/genetics , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/deficiency , Optic Atrophies, Hereditary/genetics , Optic Atrophies, Hereditary/metabolism , Actins/metabolism , Progeria/genetics , Progeria/pathology , Progeria/metabolismABSTRACT
Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.
ABSTRACT
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.
Subject(s)
Extracellular Matrix Proteins , Fraser Syndrome , Animals , Humans , Mice , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Fraser Syndrome/metabolism , Skin/metabolismABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Zebrafish , Adult , Humans , Mice , Animals , Cartilage Oligomeric Matrix Protein/genetics , Zebrafish/genetics , Phenotype , Thrombospondins/genetics , MammalsABSTRACT
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.
Subject(s)
Bone Diseases, Metabolic , Cutis Laxa , Animals , Humans , Mice , Collagen/genetics , Cutis Laxa/genetics , Elastin/metabolism , Extracellular Matrix Proteins/metabolismABSTRACT
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.
ABSTRACT
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 dermalâepidermal 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 complexâassociated 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.
Subject(s)
Epidermolysis Bullosa Dystrophica , Extracellular Matrix Proteins , Mice , Animals , Pregnancy , Female , Extracellular Matrix Proteins/metabolism , Skin/metabolism , Basement Membrane/metabolism , Epidermolysis Bullosa Dystrophica/metabolism , Collagen/metabolism , Membrane Proteins/metabolismABSTRACT
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.
Subject(s)
Extracellular Matrix , Latent TGF-beta Binding Proteins , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Fibrillin-1/genetics , Fibrillin-1/metabolism , Fibrillin-2/genetics , Fibrillin-2/metabolism , Fibrillins/metabolism , Humans , Latent TGF-beta Binding Proteins/genetics , Latent TGF-beta Binding Proteins/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Collagen Type VI/immunology , Stromal Cells/immunology , Animals , Antigens/immunology , Mice , Spleen/cytology , Spleen/immunology , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFß, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.
Subject(s)
Collagen Type XII/genetics , Skin/growth & development , Transforming Growth Factor beta/genetics , Wound Healing/genetics , Animals , Collagen Type I/genetics , Extracellular Matrix , Fibroblasts/metabolism , Fibroblasts/pathology , Homeostasis/genetics , Humans , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Knockout/genetics , Myofibroblasts/metabolism , Skin/parasitologyABSTRACT
Although collagens are the most abundant proteins implicated in various disease pathways, essential mechanisms required for their proper folding and assembly are poorly understood. Heat-shock protein 47 (HSP47), an ER-resident chaperone, was mainly reported to fulfill key functions in folding and secretion of fibrillar collagens by stabilizing pro-collagen triple-helices. In this study, we demonstrate unique functions of HSP47 for different collagen subfamilies. Our results show that HSP47 binds to the N-terminal region of procollagen I and is essential for its secretion. However, HSP47 ablation does not majorly impact collagen VI secretion, but its lateral assembly. Moreover, specific ablation of Hsp47 in murine keratinocytes revealed a new role for the transmembrane collagen XVII triple-helix formation. Incompletely folded collagen XVII C-termini protruding from isolated HSP47 null keratinocyte membrane vesicles could be fully restored upon the application of recombinant HSP47. Thus, our study expands the current view regarding the client repertoire and function of HSP47, as well as emphasizes its importance for transmembrane collagen folding.
Subject(s)
HSP47 Heat-Shock Proteins/metabolism , Keratinocytes/metabolism , Procollagen/metabolism , Protein Folding , Animals , HSP47 Heat-Shock Proteins/genetics , Mice , Procollagen/geneticsABSTRACT
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.
Subject(s)
Collagen Type VI/chemistry , Muscular Diseases , Mutation , Collagen Type VI/genetics , Crystallography, X-Ray , Humans , Protein DomainsABSTRACT
Introduction: The curricular implementation of events (or programs) for science-related training in human medicine has been on the agenda of the medical faculties since the publication of the Federal-State Working Group [1]. The Medical Faculty of the University of Cologne developed and established a systematic, longitudinal science curriculum together with the start of the model curriculum in human medicine in 2003. Here, we investigate the questions of whether the described (para-) curricular elements are accepted by students and lecturers and how they are evaluated, especially by students. In addition, we investigate whether selected parameters can be used to demonstrate changes in the students' scientific activities. Project description: The program "Research and Medical Studies" (RaMS) consists of several components: these elements of the mandatory curricular (Scientific Projects, SP) and optional components (Research in Medical Studies (RiMS), Research Track (RT), Research Fair Cologne (RFC)) are described here. Results were recorded at various levels: Likert Scale evaluation of the event's elements were collected as satisfaction parameters from the studentsProcess data on participation in the voluntary events were collected and evaluated as absolute and relational figures (WS 12/13-SS 17). Data on the outcome of the RaMS program were collected: Type of scientific projects in the academic years 2011/12-2014/15), number and type of available projects offered at the RFC (in the years 2011-18) and number of student research funding applications in a comparison of the periods 2010-13 vs. 2014-17). Results: The students' acceptance of mandatory and paracurricular courses of the RaMS program is pleasingly high, which is not surprising, at least in the case of the voluntary courses. The participation of students in RiMS, RT and RFC is satisfactory for voluntary courses. In the case of the RT, with certified participation of approximately 47% of all registrations (corresponding to 10% of the total cohort), this is comparable to similar programs. It can be shown that the number of experimental science projects has more than doubled over time in parallel with the development of RaMS. The average number of provided projects according to the RFC is 42 (which corresponds to a placement rate of approx. 1:4). The number of successful student applications for a research support grant during the period the measures were implemented has doubled. Discussion and conclusion: The RaMS program shows a route for the implementation of the SP required by the next licensing regulations in medical education, which was initially supported and expanded solitarily, later by further elements (RiMS), also in the sense of a science-based career development (RT, RFC). The student acceptance and the measured success, in the form of successful participation in the Research Track, increased choice of experimental projects, significant increase of submitted as well as approved research grants and the high project placement rate of the Research Fair, encourage the further development of the program, which is indicated in the conclusion.
Subject(s)
Program Evaluation/methods , Research/education , Curriculum/standards , Curriculum/trends , Education, Medical/methods , Education, Medical/trends , Educational Measurement/methods , Germany , Humans , Research/trendsABSTRACT
The α2δ-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ-1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.
Subject(s)
Calcium Channels, L-Type/metabolism , Gabapentin/metabolism , Thrombospondins/metabolism , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Ligands , Mass Spectrometry , Membrane Proteins/chemistry , Membrane Proteins/metabolismABSTRACT
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.
Subject(s)
Bone Morphogenetic Protein 1/metabolism , Collagen Type VI/metabolism , Proprotein Convertases/metabolism , Fibrosis , Furin/metabolism , HEK293 Cells , Humans , Insulin Resistance , Microfibrils/metabolism , Peptide Fragments/metabolism , ProteolysisABSTRACT
Inherited point mutations in collagen II in humans affecting mainly cartilage are broadly classified as chondrodysplasias. Most mutations occur in the glycine (Gly) of the Gly-X-Y repeats leading to destabilization of the triple helix. Arginine to cysteine substitutions that occur at either the X or Y position within the Gly-X-Y cause different phenotypes like Stickler syndrome and congenital spondyloepiphyseal dysplasia (SEDC). We investigated the consequences of arginine to cysteine substitutions (X or Y position within the Gly-X-Y) towards the N and C terminus of the triple helix. Protein expression and its secretion trafficking were analyzed. Substitutions R75C, R134C and R704C did not alter the thermal stability with respect to wild type; R740C and R789C proteins displayed significantly reduced melting temperatures (Tm) affecting thermal stability. Additionally, R740C and R789C were susceptible to proteases; in cell culture, R789C protein was further cleaved by matrix metalloproteinases (MMPs) resulting in expression of only a truncated fragment affecting its secretion and intracellular retention. Retention of misfolded R740C and R789C proteins triggered an ER stress response leading to apoptosis of the expressing cells. Arginine to cysteine mutations towards the C-terminus of the triple helix had a deleterious effect, whereas mutations towards the N-terminus of the triple helix (R75C and R134C) and R704C had less impact.
Subject(s)
Amino Acid Substitution , Collagen Type II/genetics , Osteochondrodysplasias/congenital , Unfolded Protein Response , Cell Line, Tumor , Cell Survival , Collagen Type II/chemistry , Collagen Type II/metabolism , HEK293 Cells , Humans , Osteochondrodysplasias/genetics , Protein Denaturation , Protein Domains , Protein Stability , Protein TransportABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Chromatography, Affinity/methods , Extracellular Matrix/metabolism , Matrilin Proteins/isolation & purification , Animals , Antibodies/immunology , Antibodies/isolation & purification , Cartilage/chemistry , Cell Culture Techniques/instrumentation , Chromatography, Affinity/instrumentation , Collagen/metabolism , Immunization/methods , Matrilin Proteins/analysis , Matrilin Proteins/chemistry , Matrilin Proteins/physiology , Mechanotransduction, Cellular , Protein Domains/physiology , Proteoglycans/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , RegenerationABSTRACT
C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.
Subject(s)
Lectins, C-Type/metabolism , Plasminogen Activators/metabolism , Tissue Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Cartilage/metabolism , Chromatography, Gel , HEK293 Cells , Humans , Immunohistochemistry , Lectins, C-Type/biosynthesis , Lectins, C-Type/genetics , Mice , Mice, Inbred C57BL , Plasminogen/metabolism , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post-translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten-dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.
Subject(s)
Dermatitis Herpetiformis/immunology , Epitopes/immunology , Transglutaminases/physiology , Animals , Apoptosis , Autoantibodies/immunology , Celiac Disease/immunology , Cell Lineage , Dermatitis Herpetiformis/enzymology , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Mutation , Phenotype , Signal Transduction , Skin/enzymology , Skin/immunology , Transglutaminases/geneticsABSTRACT
Mitochondria play a pivotal role in energy metabolism, but whether insulin signaling per se could regulate mitochondrial function has not been identified yet. To investigate whether mitochondrial function is regulated by insulin signaling, we analyzed muscle and liver of insulin receptor (IR)+/--insulin receptor substrate-1 (IRS-1)+/- double heterozygous (IR-IRS1dh) mice, a well described model for insulin resistance. IR-IRS1dh mice were studied at the age of 6 and 12 months and glucose metabolism was determined by glucose and insulin tolerance tests. Mitochondrial enzyme activities, oxygen consumption, and membrane potential were assessed using spectrophotometric, respirometric, and proton motive force analysis, respectively. IR-IRS1dh mice showed elevated serum insulin levels. Hepatic mitochondrial oxygen consumption was reduced in IR-IRS1dh animals at 12 months of age. Furthermore, 6-month-old IR-IRS1dh mice demonstrated enhanced mitochondrial respiration in skeletal muscle, but a tendency of impaired glucose tolerance. On the other hand, 12-month-old IR-IRS1dh mice showed improved glucose tolerance, but normal muscle mitochondrial function. Our data revealed that deficiency in IR/IRS-1 resulted in normal or even elevated skeletal muscle, but impaired hepatic mitochondrial function, suggesting a direct cross-talk between insulin signaling and mitochondria in the liver.