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Human cartilage model of the precocious osteoarthritis-inducing COL2A1 p.R719C reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II

ABSTRACT: 10-plex TMT dataset generated by immunoprecipitation of intracellular, epitope-tagged COL2A1 constructs along with DSP-crosslinked interactors. This experiment allows for the identification and quantitative comparison of wild-type and Arg719Cys COL2A1 interactomes in HT-1080 cells transiently transfected with COL2A1 under a constitutive promoter. Negative control samples are untransfected HT-1080 cells, used to identify high-confidence collagen-II interactors from non-specific binders.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Matthew Shoulders

PROVIDER: MSV000096151 | MassIVE | Tue Oct 22 06:09:00 BST 2024

SECONDARY ACCESSION(S): PXD057054

REPOSITORIES: MassIVE

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Project description:ObjectivesMutations in the procollagen-II gene (COL2A1) often cause chondrodysplasias, including the precocious osteoarthritis-inducing p.Arg719Cys. Understanding the molecular basis of such diseases has long been challenging, owing to a lack of models accurately reflecting disease genotypes and phenotypes. To address this challenge, we develop and characterize in vitro human cartilage derived from wild-type and disease-causing Arg719Cys COL2A1 isogenic induced pluripotent stem cell (iPSC) lines.MethodsUsing directed differentiation of iPSCs to chondrocytes, we generated cartilage from wild-type and Arg719Cys COL2A1 lines. We compared the resulting protein, cell, and tissue properties using immunohistochemistry, electron microscopy, SDS-PAGE, RNA-sequencing, and quantitative interactomics.ResultsWhile both wild-type and disease lines deposited a cartilage matrix, the Arg719Cys matrix was deficient. Arg719Cys collagen-II was excessively post-translationally modified and modestly intracellularly retained, leading to endoplasmic reticulum (ER) distention suggestive of an ER storage defect. Interactomic studies indicated that Arg719Cys procollagen-II was not differentially engaged by the ER proteostasis network. RNA-sequencing showed that the ER storage defect engendered by Arg719Cys procollagen-II also did not activate cellular stress responses, including the unfolded protein response. These data suggest that cells fail to properly recognize Arg719Cys-associated procollagen-II defects.ConclusionsA failure to identify and rectify defective procollagen-II folding in cells expressing Arg719Cys procollagen-II leads to the deposition of a sparse and defective collagen-II matrix, culminating in pathology. Combined with the highly expandable human cartilage disease model reported here, this work provides motivation and a platform to discover therapeutic strategies targeting procollagen folding, quality control, and secretion in this collagenopathy and others.

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Human cartilage model of the precocious osteoarthritis-inducing COL2A1 p.R719C reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II

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