Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

Author:

Bell Peter A.12,Wagener Raimund34,Zaucke Frank3,Koch Manuel345,Selley Julian1,Warwood Stacey1,Knight David1,Boot-Handford Raymond P.1,Thornton David J.1,Briggs Michael D.12

Affiliation:

1. Wellcome Trust Centre for CellMatrix Research, Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, UK

2. Present address: Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne NE1 3BZ, UK

3. Center for Biochemistry, University of Cologne, D50931 Cologne, Germany

4. Center for Molecular Medicine, University of Cologne, D50931 Cologne, Germany

5. Institute for Dental Research and Musculoskeletal Biology, Medical Faculty, University of Cologne, D50931 Cologne, Germany

Abstract

Summary 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.

Publisher

The Company of Biologists

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology

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