Struktur, Funktion und Expression der Matriline, einer Familie von extrazellulären Matrixproteinen
Zusammenfassung der Projektergebnisse
The matrilins form a four-member family of extracellular matrix proteins and belong to the superfamily of von Willebrand factor A (VWA)-containing proteins. Matrilins are expressed in cartilage and many other forms of extracellular matrix, where they are part of fibrillar or filamentous structures and mediate interactions between collagen-containing fibrils and other matrix components. However, gene inactivation studies in mouse were disappointing. We therefore used the zebrafish as an alternative model to study the function of the prototype member matrilin-1 in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, 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. In contrast to in mouse, knockdown of matrilin-1 in zebrafish results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of proteoglycan and collagen II deposition. The chondrocytes showed ER aberrations but no activation of XBP-1, a marker for ER 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. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the VWC 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. During the work on the structure and interactions of matrilins it became evident that expression of matrilin VWA domains is difficult due to their intrinsic tendency to aggregation. This could explain why crystallization attempts with the eukaryotically expressed matrilin-4 VWA2 and matrilin-3 VWA domains failed. Moreover we could show that bacteria are not able to properly fold matrilin VWA domains. Therefore, structure determination by NMR was not possible, as the necessary isotope labeling was not feasible. Surface plasmon resonance measurements identified matrilin-1 VWA2 as the strongest and the matrilin-2 VWA2 as the weakest binding partner for COMP among matrilin VWA domains. In an alternative approach, we crystalized the collagen VI α3 chain VWA domain N5 that is closely related to matrilin VWA domains. This structure can now serve as a template for an improved model of matrilin VWA domains. As mutations in collagen VI VWA domains lead to myopathies, the novel structure will also be helpful to model other collagen VI VWA domains and to evaluate the impact of known mutations on the domain structures, thereby providing a basis for genotype phenotype analysis of collagen VI related myopathies.
Projektbezogene Publikationen (Auswahl)
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2007. Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice. J. Biol. Chem. 282, 22163-75
Nicolae, C., Ko, Y.P., Miosge, N., Niehoff, A., Studer, D., Enggist, L., Hunziker, E.B., Paulsson, M., Wagener, R., Aszodi, A.
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2007. Matrilins mediate weak cell attachment without promoting focal adhesion formation. Matrix Biol. 26, 167-74
Mann, H.H., Sengle, G., Gebauer, J.M., Eble, J.A., Paulsson, M., Wagener, R.
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2008. Three novel collagen VI chains with high homology to the alpha 3 chain. J. Biol. Chem. 283, 10658-70
Gara, S.K., Grumati, P., Urciuolo, A., Bonaldo, P., Kobbe, B., Koch, M., Paulsson, M., Wagener, R.
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2009. Matrilin-3 activates the 1 expression of osteoarthritis-associated genes in primary human chondrocytes. FEBS Lett. 583, 3611-7
Klatt, A.R., Klinger, G., Paul-Klausch, B., Kühn, G., Renno, J.H., Wagener. R., Paulsson, M., Schmidt, J., Malchau, G., Wielckens, K.
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2009. Proteolytic processing causes extensive heterogeneity of tissue matrilin forms. J. Biol. Chem., 284, 21545-56
Ehlen, H.W., Sengle, G., Klatt, A.R., Talke, A., Müller, S., Paulsson, M., Wagener, R.
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The extracellular-matrix protein matrilin 2 participates in peripheral nerve regeneration. 2009. J Cell Sci. 122, 995-1004
Malin, D., Sonnenberg-Riethmacher, E., Guseva, D., Wagener, R., Aszódi, A., Irintchev, A., Riethmacher, D.
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2011.The matrilins: modulators of extracellular matrix assembly. Int. J. Biochem. Cell Biol. 43, 320-30
Klatt, A.R, Becker, A.K., Neacsu, C.D., Paulsson, M., Wagener, R.
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2013. The matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes. Osteoarthritis Cartilage. 21, 869-73
Klatt AR, Paul-Klausch B, Klinger G, Hillebrand U, Kühn G, Kobbe B, Renno JH, Johannis W, Paulsson M, Wagener R
