Genotype-phenotype relations in patient-derived point mutations in collagen II

Collagen II is the major collagen present in the extracellular matrix of cartilage, in addition it is found in the vitreous of the eye and it is also detected during early embryogenesis. Due to the complexity of the biosynthesis, assembly and secretion, collagen II is highly susceptible to mutations leading to disease states which are broadly classified as chondrodysplasias. Most of these mutations are substitutions of glycine in the Gly-X-Y repeats in the triple helical domain resulting in destabilization of the helix. Point mutations leading to arginine to cysteine substitution are interesting since they occur at either X or Y position and cause two different diseases termed Stickler syndrome and congenital spondyloepiphyseal dysplasia (SEDC) in association with osteoarthritis. The present study aimed at determining the consequences of arginine to cysteine substitutions in either X or the Y position in the Gly-X-Y repeats and at either the N- or the C- terminus of the triple helix. The impact of these mutations on protein trafficking, secretion and cell survival was also analyzed. Biochemical studies revealed great similarities between R75C, R134C and R704C collagens and the wild type molecules with the exception that electron micrographs of R75C collagen displayed kinks in the structure. R740C and R789C collagens accumulated in the cells and the R789C protein migrated faster on SDS gels. The R740C and R789C proteins were also susceptible to protease digestion and circular dichroism spectra were altered and showed lower Tm values than other collagen II variants. Due to the altered structure, R789C protein was more susceptible to MMP cleavage in the vicinity of the mutation causing the truncation of the protein. Additionally, electron micrographs revealed only scarce and thin filamentous structures in preparations of R740C and R789C protein. The biochemical results indicate that the R740C and R789C proteins have unstable triple helices and that this affects the overall protein structure. Protein trafficking was monitored in HT1080 cells expressing the collagen II variants. Intracellular retention in the ER due to misfolding of the R740C and R789C proteins triggered an ER stress response including splicing of XBP-1 and induction and binding of BiP. Continuous accumulation of misfolded proteins in the ER caused apoptosis of the R740C and R789C expressing cells. Substitution of arginine to cysteine in the X or Y position towards the C-terminus of the triple helix caused pronounced instability of the triple helix with a deleterious effect on the cells, while R704C and more N-terminal mutations did not cause any significant changes irrespective of being in the X or the Y position. The different severities of patient phenotypes are due to a combination of structural factors, which may be synergistically augmented by genetic modifiers, additional unknown mutations and environmental factors