Structural and Functional Investigations of Matrilin-1 A-domains Reveal Insights into Their Role in Cartilage ECM Assembly*

Abstract

Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn2+ suggesting that matrilin-3 has acquired a requirement for specific metal ions for activation and/or molecular associations. To understand the interdependence of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability, and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn2+ also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II·VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome

“…A third protein band, which was clearly and consistently reduced in the collagen null P3 E1 extracts, was identified as matrilin-1. This is consistent with the interaction between matrilin-1 A-domains and collagen IX shown in vitro (36) and provides the first evidence for the association of collagen IX and matrilin-1 under native conditions in vivo.…”

Loss of matrilin 1 does not exacerbate the skeletal phenotype in a mouse model of multiple epiphyseal dysplasia caused by a Matn3 V194D mutation

“…MED is genetically heterogeneous, and autosomal‐dominant forms of the disease arise from mutations in the genes encoding matrilin 3, cartilage oligomeric matrix protein (COMP), and type IX collagen (3–5). Although present in other musculoskeletal tissues, matrilin 3, COMP, and type IX collagen are expressed primarily by chondrocytes and have been shown to interact with each other, both in vitro and in vivo (6–13).…”

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

“…In the first instance we used a candidate approach to determine the extractability of a number of ECM molecules including members of the matrilin protein family, type IX collagen and decorin, which are all known to interact with matrilin-3 and COMP (Budde et al, 2005; Fresquet et al, 2010; Fresquet et al, 2007; Holden et al, 2001; Mann et al, 2004; Wiberg et al, 2003). Articular and epiphyseal cartilage from 3-week-old mouse knee joints was sequentially extracted in a series of three buffers and the extracted proteins were separated by SDS-PAGE and visualised by Western blotting.…”

“…SDS-PAGE and Western blot analysis of sequentially extracted knee cartilage revealed genotype-specific differences in the extraction of a number of proteins that included matrilin-1 to -4, COMP and type IX collagen, which are all known to interact with each other (Budde et al, 2005; Fresquet et al, 2010; Fresquet et al, 2007; Holden et al, 2001; Mann et al, 2004; Wiberg et al, 2003). While we were unable to analyse the insoluble material that remained following extraction of cartilage tissue (and therefore could not quantify total differences in protein abundance), our method was especially useful in allowing us to identify subtle differences in the extraction of individual protein oligomers that may not have been detected using other methods.…”