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Raimund Wagener - One of the best experts on this subject based on the ideXlab platform.
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Characterization of recombinantly expressed Matrilin VWA domains.
Protein expression and purification, 2014Co-Authors: Ann-kathrin A. Becker, Mats Paulsson, Halina Mikolajek, Jörn M. Werner, Raimund WagenerAbstract: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.
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Matrilin-1 is Essential for Zebrafish Development by Facilitating Collagen II Secretion
The Journal of biological chemistry, 2013Co-Authors: Cristian Dan Neacsu, Mats Paulsson, Andreas Tagariello, Kristina Røkenes Karlsen, Wolfram F. Neiss, Raimund WagenerAbstract:Matrilin-1 is the prototypical member of the Matrilin protein family and is highly expressed in cartilage. However, gene targeting of Matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study Matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, Matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, Matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of Matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the Matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of Matrilin-1 deficiency. The results show that Matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.
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Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures.
Biology open, 2013Co-Authors: Peter A. Bell, Frank Zaucke, Ray Boot-handford, Raimund Wagener, Julian N. Selley, Stacey Warwood, David Knight, David J ThorntonAbstract:Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of Matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of Matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of Matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state.
Matrix biology : journal of the International Society for Matrix Biology, 2011Co-Authors: Gergely Groma, Mats Paulsson, Raimund Wagener, Attila Aszódi, Harald W. A. Ehlen, Ivan Grskovic, Bent Brachvogel, Sylvia Schael, Amanda J. Fosang, Frank ZauckeAbstract:The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of Matrilins in the secretory pathway. The Matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed Matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of Matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a Matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved Matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The Matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of Matrilins and potentially of other extracellular matrix proteins.
Mats Paulsson - One of the best experts on this subject based on the ideXlab platform.
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Characterization of recombinantly expressed Matrilin VWA domains.
Protein expression and purification, 2014Co-Authors: Ann-kathrin A. Becker, Mats Paulsson, Halina Mikolajek, Jörn M. Werner, Raimund WagenerAbstract: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.
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Matrilin-1 is Essential for Zebrafish Development by Facilitating Collagen II Secretion
The Journal of biological chemistry, 2013Co-Authors: Cristian Dan Neacsu, Mats Paulsson, Andreas Tagariello, Kristina Røkenes Karlsen, Wolfram F. Neiss, Raimund WagenerAbstract:Matrilin-1 is the prototypical member of the Matrilin protein family and is highly expressed in cartilage. However, gene targeting of Matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study Matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, Matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, Matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of Matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the Matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of Matrilin-1 deficiency. The results show that Matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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Abnormal bone quality in cartilage oligomeric matrix protein and Matrilin 3 double-deficient mice caused by increased tissue inhibitor of metalloproteinases 3 deposition and delayed aggrecan degradation.
Arthritis and rheumatism, 2012Co-Authors: Gergely Groma, Mats Paulsson, Anja Niehoff, Wei Xin, Ivan Grskovic, Bent Brachvogel, Frank ZauckeAbstract:Objective Cartilage oligomeric matrix protein (COMP) and Matrilin 3 are extracellular matrix proteins that are abundant in cartilage. As adaptor molecules, both proteins bridge and stabilize macromolecular networks consisting of fibrillar collagens and proteoglycans. Mutations in the genes coding for COMP and Matrilin 3 have been linked to human chondrodysplasias, while in mice, deficiency in COMP or Matrilin 3 does not cause any pronounced skeletal abnormalities. Given the similar functions of COMP and Matrilin 3 in the assembly and stabilization of the extracellular matrix, our aim was to determine whether these proteins could functionally compensate for each other. Methods To assess this putative redundancy of COMP and Matrilin 3, we generated COMP/Matrilin 3 double-deficient mice and performed an in-depth analysis of their skeletal development. Results At the newborn stage, the overall skeletal morphology of the double mutants was normal, but at 1 month of age, the long bones were shortened and the total body length reduced. Peripheral quantitative computed tomography revealed increased metaphyseal trabecular bone mineral density in the femora. Moreover, the degradation of aggrecan in the cartilage remnants in the metaphyseal trabecular bone was delayed, paralleled by increased deposition of tissue inhibitor of metalloproteinases 3 (TIMP-3). The structure and morphology of the growth plate were grossly normal, but in the center, focal closures were observed, a phenotype very similar to that described in matrix metalloproteinase 13 (MMP-13)–deficient mice. Conclusion We propose that a lack of COMP and Matrilin 3 leads to increased deposition of TIMP-3, which causes partial inactivation of MMPs, including MMP-13, a mechanism that would explain the similarities in phenotype between COMP/Matrilin 3 double-deficient and MMP-13–deficient mice.
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Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state.
Matrix biology : journal of the International Society for Matrix Biology, 2011Co-Authors: Gergely Groma, Mats Paulsson, Raimund Wagener, Attila Aszódi, Harald W. A. Ehlen, Ivan Grskovic, Bent Brachvogel, Sylvia Schael, Amanda J. Fosang, Frank ZauckeAbstract:The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of Matrilins in the secretory pathway. The Matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed Matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of Matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a Matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved Matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The Matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of Matrilins and potentially of other extracellular matrix proteins.
Birgit Kobbe - One of the best experts on this subject based on the ideXlab platform.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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the Matrilins adaptor proteins in the extracellular matrix
FEBS Letters, 2005Co-Authors: Raimund Wagener, Birgit Kobbe, Harald W. A. Ehlen, Henning H. Mann, Gerhard Sengle, Mats PaulssonAbstract:The Matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study Matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in Matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of Matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.
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The Matrilins – adaptor proteins in the extracellular matrix
FEBS letters, 2005Co-Authors: Raimund Wagener, Birgit Kobbe, Harald W. A. Ehlen, Henning H. Mann, Gerhard Sengle, Mats PaulssonAbstract:The Matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study Matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in Matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of Matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.
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Zebrafish (Danio rerio) Matrilins: shared and divergent characteristics with their mammalian counterparts.
The Biochemical journal, 2005Co-Authors: Birgit Kobbe, Mats Paulsson, Raimund WagenerAbstract:We have cloned the cDNAs of the zebrafish (Danio rerio) members of the Matrilin family of extracellular adaptor proteins. In contrast to mammals, no orthologue of Matrilin-2 was found in zebrafish, either by RT (reverse-transcriptase) PCR using degenerated primers or by screening the databases (Ensembl and NCBI); however, two forms of Matrilin-3, Matrilin-3a and -3b, were present. The identity with the mammalian Matrilins is from more than 70% for the VWA (von Willebrand factor A)-like domains to only 28% for the coiled-coil domains of Matrilin-3a and -3b. In all zebrafish Matrilins we found a greater variety of splice variants than in mammals, with splicing mainly affecting the number of EGF (epidermal growth factor)-like repeats. The exon-intron organization is nearly identical with that of mammals, and also the characteristic AT-AC intron interrupting the exons coding for the coiled-coil domain is conserved. In the Matrilin-3b gene a unique exon codes for a proline- and serine/threonine-rich domain, possibly having mucin-like properties. The Matrilin-1 and -3a genes were mapped to chromosome 19 and 20 respectively by the radiation hybrid method. The temporal and spatial expression of zebrafish Matrilins is similar to that seen in the mouse. Zebrafish Matrilin-4 is highly expressed as early as 24 hpf (h post fertilization), whereas the other Matrilins show peak expression at 72 hpf. By immunostaining of whole mounts and sections, we found that Matrilin-1 and -3a show predominantly skeletal staining, whereas Matrilin-4 is more widespread, with the protein also being present in loose connective tissues and epithelia.
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Zebrafish (Danio rerio) Matrilins: shared and divergent characteristics with their mammalian counterparts
Biochemical Journal, 2005Co-Authors: Birgit Kobbe, Mats Paulsson, Raimund WagenerAbstract:We have cloned the cDNAs of the zebrafish (Danio rerio) members of the Matrilin family of extracellular adaptor proteins. In contrast to mammals, no orthologue of Matrilin-2 was found in zebrafish, either by RT (reverse-transcriptase) PCR using degenerated primers or by screeening the databases (Ensembl and NCBI); however, two forms of Matrilin-3, Matrilin-3a and -3b, were present. The identity with the mammalian Matrilins is from more than 70% for the VWA (von Willebrand factor A)-like domains to only 28% for the coiled-coil domains of Matrilin-3a and -3b. In all zebrafish Matrilins we found a greater variety of splice variants than in mammals, with splicing mainly affecting the number of EGF (epidermal growth factor)-like repeats. The exon–intron organization is nearly identical with that of mammals, and also the characteristic AT–AC intron interrupting the exons coding for the coiled-coil domain is conserved. In the Matrilin-3b gene a unique exon codes for a proline- and serine/threonine-rich domain, possibly having mucin-like properties. The Matrilin-1 and -3a genes were mapped to chomosome 19 and 20 respectively by the radiation hybrid method. The temporal and spatial expression of zebrafish Matrilins is similar to that seen in the mouse. Zebrafish Matrilin-4 is highly expressed as early as 24 hpf (h post fertilization), whereas the other Matrilins show peak expression at 72 hpf. By immunostaining of whole mounts and sections, we found that Matrilin-1 and -3a show predominantly skeletal staining, whereas Matrilin-4 is more widespread, with the protein also being present in loose connective tissues and epithelia.
Andreas R Klatt - One of the best experts on this subject based on the ideXlab platform.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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The Matrilins: modulators of extracellular matrix assembly.
The international journal of biochemistry & cell biology, 2010Co-Authors: Andreas R Klatt, Mats Paulsson, Ann-kathrin A. Becker, Cristian Dan Neacsu, Raimund WagenerAbstract:The Matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a Matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in Matrilin structure may lead to a modulation of extracellular matrix assembly. Some Matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though Matrilins play a role in mechanotransduction and Matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of Matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of Matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of Matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on Matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.
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Matrilin 3 activates the expression of osteoarthritis associated genes in primary human chondrocytes
FEBS Letters, 2009Co-Authors: Andreas R Klatt, Mats Paulsson, Raimund Wagener, Gabriele Klinger, Gertrud Kühn, Joerg H. Renno, Joachim Schmidt, Gebhart Malchau, Brigitte Paulklausch, Klaus WielckensAbstract:Here, we tested the Matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1β, TNFα, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of Matrilin-3. We provide evidence for a Matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released Matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1β. ADAMTS4 and ADAMTS5, in turn, cleave Matrilin-3 and may release more Matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.
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Matrilin‐3 activates the expression of osteoarthritis‐associated genes in primary human chondrocytes
FEBS letters, 2009Co-Authors: Andreas R Klatt, Mats Paulsson, Raimund Wagener, Brigitte Paul-klausch, Gabriele Klinger, Gertrud Kühn, Joerg H. Renno, Joachim Schmidt, Gebhart Malchau, Klaus WielckensAbstract:Here, we tested the Matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1β, TNFα, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of Matrilin-3. We provide evidence for a Matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released Matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1β. ADAMTS4 and ADAMTS5, in turn, cleave Matrilin-3 and may release more Matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.
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Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms
The Journal of biological chemistry, 2009Co-Authors: Harald W. A. Ehlen, Mats Paulsson, Andreas R Klatt, Anja Talke, Gerhard Sengle, Stefan Müller, Raimund WagenerAbstract:The Matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of Matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the Matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in Matrilin-1 and particularly complex in Matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in Matrilin-4 by the Matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that Matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not Matrilin-1 subunits present in Matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any Matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of Matrilin-4 detected processed Matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.
Rikard Holmdahl - One of the best experts on this subject based on the ideXlab platform.
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Relapsing polychondritis, induced in mice with Matrilin 1, is an antibody- and complement-dependent disease.
The American journal of pathology, 2004Co-Authors: Ann-sofie Hansson, Dick Heinegård, Martina Johannesson, Lars Svensson, Kutty Selda Nandakumar, Rikard HolmdahlAbstract:Relapsing polychondritis is an autoimmune disease that affects cartilage in the ear, nose, and respiratory tract. A pathogenic immune response has been proposed and antibodies to several cartilage proteins are detected in sera from these patients. To investigate the role of the humoral immune response in relapsing polychondritis, we used the Matrilin-1-induced relapsing polychondritis model. Mice deficient of B cells (μMT) and mice congenic at the complement factor 5, were immunized with Matrilin-1, a cartilage-specific protein mainly detected in the tracheal cartilage. To investigate the binding properties and tissue selection of Matrilin-1-specific antibodies we produced Matrilin-1-specific B-cell hybridomas. Although 83% of the μMT heterozygous mice developed respiratory distress and erosive chondritis in the respiratory tract, none of the B-cell-deficient mice were susceptible to disease. In addition, we show that complement factor 5 is important for the induction of Matrilin-1-induced relapsing polychondritis. Monoclonal Matrilin-1-specific antibodies injected into neonatal mice bound specifically to cartilage of the respiratory tract and adult B-cell-deficient mice injected with the same antibodies developed erosive chondritis in the respiratory tract. We conclude that relapsing polychondritis can be mediated by a pathway involving tissue-specific antibodies and complement activation.
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The occurrence of autoantibodies to Matrilin 1 reflects a tissue-specific response to cartilage of the respiratory tract in patients with relapsing polychondritis.
Arthritis and rheumatism, 2001Co-Authors: Ann-sofie Hansson, Dick Heinegård, Jean-charles Piette, Harald Burkhardt, Rikard HolmdahlAbstract:Objective Relapsing polychondritis (RP) is an inflammatory disease that mainly affects cartilage tissue in the auricle, nose, and lower respiratory tract. When tracheolaryngeal cartilage is involved, the disease is occasionally fatal. Matrilin 1 is a cartilage-specific protein most prominently expressed in tracheal cartilage, but not in joint cartilage. Immunization with the protein in rats and mice induces respiratory distress and nasal destruction, as seen in RP. We investigated the response to Matrilin 1 and other cartilage proteins in sera from patients with RP, 4 additional groups of patients with other major connective tissue diseases, and healthy control subjects. Methods Sera were analyzed by enzyme-linked immunosorbent assay (ELISA) for antibody responses to Matrilin 1, types II, IX, and XI collagen, and cartilage oligomeric matrix protein (COMP). Titers above the mean + 3SD of controls were considered positive. Specificity of Matrilin 1 recognition was further investigated by the capacity of high-titer sera to block the binding of a Matrilin 1–specific monoclonal antibody in inhibition ELISAs. In vivo reactivity and specificity were tested by injecting sera into neonatal mice, and antibody binding was detected by immunohistochemical staining. Results Serum antibodies from RP patients bound tracheolaryngeal and nasal cartilage in vivo and inhibited the binding of anti–Matrilin 1–specific monoclonal antibodies. Thirteen of the 97 RP patients had increased titers of Matrilin 1 antibody. Positive titers correlated with respiratory symptoms in 69% of the cases. Significant responses to type II collagen and COMP were also detected. Conclusion Antibodies to Matrilin 1 bind tracheolaryngeal cartilage in vivo and are correlated with an inflammatory attack on tracheolaryngeal cartilage that is often seen in RP.