Misfolding of Collagen X Chains Harboring Schmid Metaphyseal Chondrodysplasia Mutations Results in Aberrant Disulfide Bond Formation, Intracellular Retention, and Activation of the Unfolded Protein Response

Abstract

Collagen X is a short chain collagen expressed specifically by the hypertrophic chondrocytes of the cartilage growth plate during endochondral bone formation. Accordingly, COL10A1 mutations disrupt growth plate function and cause Schmid metaphyseal chondrodysplasia (SMCD). SMCD mutations are almost exclusively located in the NC1 domain, which is crucial for both trimer formation and extracellular assembly. Several mutations are expected to reduce the level of functional collagen X due to NC1 domain misfolding or exclusion from stable trimer formation. However, other mutations may be tolerated within the structure of the assembled NC1 trimer, allowing mutant chains to exert a dominant-negative impact within the extracellular matrix. To address this, we engineered SMCD mutations that are predicted either to prohibit subunit folding and assembly (NC1del10 and Y598D, respectively) or to allow trimerization (N617K and G618V) and transfected these constructs into 293-EBNA and SaOS-2 cells. Although expected to form stable trimers, G618V and N617K chains (like Y598D and NC1del10 chains) were secreted very poorly compared with wild-type collagen X. Interestingly, all mutations resulted in formation of an unusual SDS-stable dimer, which dissociated upon reduction. As the NC1 domain sulfhydryl group is not solventexposed in the correctly folded NC1 monomer, disulfide bond formation would result only from a dramatic conformational change. In cells expressing mutant collagen X, we detected significantly increased amounts of the spliced form of X-box DNA-binding protein mRNA and up-regulation of BiP, two key markers for the unfolded protein response. Our data provide the first clear evidence for misfolding of SMCD collagen X mutants, and we propose that solvent exposure of the NC1 thiol may trigger the recognition and degradation of mutant collagen X chains.Collagen X is a major constituent of the pericellular matrix of hypertrophic chondrocytes within the cartilage growth plate (1), and the expression of collagen X during endochondral ossification is intimately linked to the onset of cartilage calcification and extracellular matrix remodeling (2). The absence of a functional collagen X network in mice is associated with displacement of proteoglycans, altered mineral deposition, compression of the growth plate, and hematopoietic changes (3-5). Based on these studies, it has been proposed that collagen X interactions within the cartilage extracellular matrix establish the correct microenvironment for matrix mineralization and subsequent bone development.The human ␣1(X) collagen chain has a short collagenous domain flanked at the C and N termini by globular NC1 and NC2 domains. The NC1 domain plays a critical role in intracellular trimer formation (6, 7) and also contributes to the stability of extracellular collagen X networks (8, 9), probably involving aromatic residues exposed on the surface of each NC1 subunit (10). Mutations in the COL10A1 gene result in Schmid metaphyseal chondrodysplasia (SMCD), 1 an autosomal dominant ...

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