Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta, aka OI). However, these mutations do not elicit the expected Endoplasmic Reticulum (ER) stress response, in contrast to other protein folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response stress nor ER overload. Despite pronounced ER dilation, there is no upregulation of BIP expected in the former and no activation of NFκB signaling expected in the latter. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation of autophagy, upregulation of general stress response protein CHOP, and osteoblast malfunction reveal some other adaptive response to the ER disruption. We show how this response alters differentiation and function of osteoblasts in culture and in vivo. We demonstrate that bone matrix deposition by cultured osteoblasts is rescued by activation of misfolded procollagen autophagy, suggesting a new therapeutic strategy for OI.

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (⌬T m ) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of ⌬T m with the identity of the substituting residue. Instead, we observed regular variations in ⌬T m with the substitution location in different triple helix regions. To relate the ⌬T m map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (⌬G ‡ ) from the reported peptide data. We constructed the ⌬G ‡ map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the ⌬T m and ⌬G ‡ maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the ⌬T m map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the ␣1(I) chain.The mature type I collagen molecule is a 300-nm-long triple helix formed by two ␣1(I) and one ␣2(I) chains, which is flanked by short terminal peptides. Based on the (Gly-Xaa-Yaa) 338 triplet repeat within each chain (1), the triple helix is commonly viewed as a single domain (Xaa and Yaa stand for variable residues). However, this picture may not accurately represent variations in the stability of different regions within the triple helix that fold and unfold cooperatively (2-4). The triple helix is only metastable or marginally stable at physiological temperature (3, 5-8). Its local structure appears to be highly dynamic and intimately related to local stability. The more labile regions may exist in a loose conformation, constantly undergoing unfolding/refolding transitions while more stable "clamp" regions prevent unfolding of the whole molecule (3, 9 -12). Such structural and dynamic heterogeneity is believed to play an important role in self-assembly (9, 13) and function (3) of collagen fibers.Significant progress in understanding regional variations in the triple helix stability in different collagens has been reported in recent years. For example, some flexible sites were localized by observing triple helix bending in electron microscopy (14) and/or increased susceptibility to proteolytic cleavage (9, 15). Genetically generated reshuffling of different triple helix regions was shown to have a significant effect on the overall stability of the molecule (11, 16). Relative local stability maps were proposed based on scoring different sequences (17) or on the denaturation temperature (T m ) measured for triple-helical host-guest peptides (18).The existence of looser, less stable, and tighter, more stable structural regions within the triple helix is now commonly accepted. However, knowledge of their loca...

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.