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

Peter A Bell,Raymond P Boot-Handford,Julian Selley,David J Thornton,David Knight,Michael D Briggs,Stacey Warwood,Frank Zaucke,Raimund Wagener,Manuel Koch

doi:10.1242/bio.20135280

Abstract

SummaryPseudoachondroplasia 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.

Highlights

The chondrodysplasias are a diverse group of many different phenotypes that arise when endochondral bone growth is disrupted and include two clinically related phenotypes; multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH) (Warman et al, 2011)
We demonstrated that mutation of matrilin-3 and cartilage oligomeric matrix protein (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
This study describes for the first time the proteomic characterisation of cartilage from Matn3 V194D, Comp T585M and Comp DelD469 mutant mice using a combination of Western blotting and semi-quantitative spectral counting and identifies both common and unique disease signatures that provide insight into detrimental changes that may predispose to cartilage degeneration

Summary

Introduction

The chondrodysplasias are a diverse group of many different phenotypes that arise when endochondral bone growth is disrupted and include two clinically related phenotypes; multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH) (Warman et al, 2011). MED is genetically heterogeneous and can be caused by mutations in genes encoding the extracellular matrix (ECM) proteins matrilin-3, type IX collagen and cartilage oligomeric matrix protein (COMP) (Briggs and Chapman, 2002; Jackson et al, 2012); PSACH results exclusively from COMP mutations (Jackson et al, 2012) and is more severe than MED, but both phenotypes comprise a disease spectrum with symptoms that can include joint pain and stiffness, lower-limb deformities and early onset osteoarthritis (Briggs and Chapman, 2002). We have previously generated targeted mouse models of PSACH-MED with mutations in matrilin-3 (moderate MED: Matn V194D) and COMP (mild PSACH: Comp T585M and severe PSACH: Comp DelD469) and described the resulting phenotypes in detail (Leighton et al, 2007; Pirog-Garcia et al, 2007; Suleman et al, 2012).

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