Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756800 |
| Date | January 2015 |
| Creators | Edwards, Sarah |
| Contributors | Boot-Handford, Raymond |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-role-of-a-novel-er-molecular-chaperone-creld2-in-the-physiology-and-pathophysiology-of-endochondral-bone-growth(6fd49909-beec-42d1-a546-8b2411616e59).html |
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