Structural and Functional Characterization of Recombinant Matrilin-3 A-domain and Implications for Human Genetic Bone Diseases

Maryline Fresquet,Thomas A Jowitt,Joni Ylöstalo,Paul Coffey,Roger S Meadows,Leena Ala-Kokko,David J Thornton,Michael D Briggs

doi:10.1074/jbc.m705301200

Abstract

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.

Highlights

Matrilin-3 is a heteromeric extracellular matrix (ECM)2 protein (50 kDa) that is primarily expressed in the cartilage growth plate [1, 2]
It has been hypothesized that A-domains, which contain a perfect metal iondependent adhesion site (MIDAS) motif, may bind a metal ion and undergo an “integrin switch” [24]
In this study DPI analysis confirmed that Zn2ϩ binding to the matrilin-3 A-domain induces a tightening of the molecule, which is equivalent to an ϳ0.3 nm decrease in hydrodynamic radius when assayed by sedimentation analysis

Summary

Introduction

Matrilin-3 is a heteromeric extracellular matrix (ECM)2 protein (50 kDa) that is primarily expressed in the cartilage growth plate [1, 2]. To study the effect of different cations on the binding properties of the matrilin-3 A-domain, type II collagen (10 ␮g/ml) was diluted in Tris-buffered saline buffer containing 1 mM of either Zn2ϩ, Ca2ϩ, Mg2ϩ, or Mn2ϩ and was injected over the chip for 3 min at 20 ␮l/min. These data demonstrated that type II collagen could bind to the matrilin-3 A-domain over a broad range of Zn2ϩ concentrations.

Results

Conclusion