Comparative proteomic analysis of normal mouse cartilage and cartilage lacking collagen IX: Altered extracellular matrix composition and interactions

The cartilage ECM is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3 and COMP is essential for cartilage matrix stability, as multiple epiphyseal dysplasia patients with Col9a1, Col9a2, Col9a3, Comp and Matn3 mutations develop early onset osteoarthritis. Ablation of COMP and matrilin-3 expression in mice results in no overt phenotype, while the collagen IX knockout has severely disturbed growth plate organization, hypocellular regions and abnormal chondrocyte shape. This abnormal chondrocyte differentiation is likely to involve altered cell-matrix interactions via an unknown mechanism. To further investigate the role of collagen IX and molecular basis of the collagen IX-/- phenotype we quantified protein expression differences between wild-type and knockout femoral head cartilage at the global level by nanoLC tandem mass spectrometry (LTQ-Orbitrap), using 2-D electrophoresis and Western blotting to validate our results. Additional analysis was performed on COMP -/- cartilage to ascertain differences related specifically to the collagen IX phenotype. We identified 403 and 472 proteins (≥2 unique peptides) by nanoLC-MS/MS analysis of 3-day (P3) and 21-day (P21) cartilage, respectively. Collagen IX ablation was associated with dramatically reduced levels of COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan and thrombospondin-4 levels were significantly reduced in P3 and P21 collagen IX-/- cartilage, providing the first in vivo evidence for these proteins as members of the collagen IX “interactome”. Changes in the levels of TGFb-induced protein igh3 and fibronectin were specifically associated with collagen IX ablation and not detected in COMP -/- cartilage, suggesting a specific role in the collagen IX phenotype. This first differential analysis of wild-type and mutant mouse cartilage demonstrates the feasibility for identification of novel protein interactions in vivo, leading the way for further analysis of mutant cartilage lacking matrix components or harbouring disease-causing mutations.