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

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 ␤-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. Matrilin-3 is a heteromeric extracellular matrix (ECM)2 protein (50 kDa) that is primarily expressed in the cartilage growth plate (1, 2). Matrilin-3 has a modular structure comprising a von Willebrand factor A domain (A-domain), four epidermal growth factor-like domains, and a coiled-coil domain, which facilitates oligomerization (1-4). Mutations in the A-domain of matrilin-3 have been shown to result in some forms of multiple epiphyseal dysplasia (MED), an autosomal dominant skeletal dysplasia characterized by short-limb dwarfism and early onset osteoarthritis (5, 6). The majority of MATN3 mutations that cause MED are missense mutations found within the ␤-strands and are predicted to affect the folding of the A-domain (6, 7). Previous in vitro and in vivo studies have shown that mutant matrilin-3 protein is retained within the rough endoplasmic reticulum of cells and elicits an unfolded protein response (7-9). This essentially renders biochemical and biophysical analysis of the effects of the mutations very difficult, because it is not possible to isolate these recombinant mutant proteins under non-denaturing conditions. MED is genetically heterogeneous and can also result in mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A1, COL9A2, and COL9A3) (reviewed in Ref. 10). COMP is the fifth member of the thrombos...

Structural and functional features of a collagen-binding matrix protein from the mussel byssus

“…It seems feasible that zinc ions have a non-specific stimulating effect on electrostatic interactions between PTMP1 and collagen. Similar effects were observed for another collagen-binding VWA domain protein, Matrilin 3, with collagen-binding properties being also enhanced but not strictly dependent on zinc ions 13 .…”

“…It has previously been hypothesized that PTMP1, like other VWA domain containing proteins, binds with and interconnects byssal collagens 9,11 . Due to the lack of available PreCol proteins, we analysed binding of heterologous collagens to PTMP1 and its isolated VWA domains using surface plasmon resonance spectroscopy (SPR) 13,27,28 , since all fibrous collagens show similar structural features. PTMP1 bound immobilized collagen type I with apparent high affinity in the sub-micromolar range as shown by the difference-sensorgram of the collagen-coated versus the reference cell (Fig.…”

“…To analyse a putative dependence of collagen-binding on metal ions that are important in case of integrins 14 or matrilins 13 but not in case of the von Willebrand factor 30 , the influence of Ca 2 þ , Mg 2 þ , Mn 2 þ or Zn 2 þ was tested. The selective binding of Zn 2 þ to the A1 MIDAS motif indicates a contribution to collagen binding.…”

“…It revealed two sequence stretches with homology to the group of von Willebrand factor type A (VWA) domains (Pfam PF00092) with B25% sequence identity andB50% homology to other members of this group, including the von Willebrand factor A3 domain or the I-domains of integrins. VWA domain containing proteins of the blood plasma or the extracellular matrix are often involved in protein-protein interactions including binding of collagen [12][13][14][15] . In case of the von Willebrand factor, binding to partner proteins like glycoprotein Ib or collagen is dependent on conformational changes upon shear forces 16,17 .…”

Novel mutations in exon 2 ofMATN3affect residues within the α-helices of the A-domain and can result in the intracellular retention of mutant matrilin-3

“…Matrilin-3 can form heterooligomers with matrilin-1 (Wu and Eyre, 1998;Zhang and Chen, 2000) and has been shown to bind to COMP and type II and IX collagen in vitro (Budde, et al, 2005;Mann, et al, 2004). More recently the A-domain of matrilin-3 has been shown to interact with the COL3 domain of type IX collagen and this interaction is abolished when type IX collagen harbors an in-frame deletion that is comparable to that caused by the skipping of exon 3 of COL9A3 (Fresquet et al, 2007); a mutation shown to cause MED (Bonnemann, et al, 2000;Lohiniva, et al, 2000;Paassilta, et al, 1999). These latter studies provide the first biochemical insight into why MED mutations might cluster in the COL3 domain of type IX collagen and provide a rationale for the non-allelic genetic heterogeneity seen in MED.…”