Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

Abstract

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.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

“…Thus, slower migration of the ␣1(I), ␣2(I), and ␤ chains of collagen type I is often detected by SDS-PAGE analysis in cultured fibroblasts from OI patients with mutations in COL1A1, COL1A2, CRTAP, LEPRE1, or PPIB (25)(26)(27)(28). In the human HSP47 OI case, migration of cell culture collagen was reported to be normal.…”

“…This suggests that the overhydroxylated type I collagen in the extracellular matrix might disturb the bone-specific cross-linking of type I collagen and negatively influence trabecular bone formation, thereby contributing to the OI pathology. Abnormal collagen cross-linking is characteristic of several subtypes of OI including Bruck syndrome, in which bone collagen lacks pyridinolines, and subtypes of the Ehlers-Danlos syndrome; in both of these conditions, the diagnosis can be confirmed by analysis of pyridinoline cross-links in urine (28,30,(32)(33)(34)(35).…”

Collagen Type I as a Ligand for Receptor-Mediated Signaling

“…Mutations in CRTAP, P3H1/LEPRE1, or PPIB (the gene that encodes cyclophilin B) strongly affect post-translational modifications of collagen resulting in a complete absence of proline 3-hydroxylation in the case of mutations in CRTAP, P3H1, and site-specific alterations in the hydroxylation and glycosylation of collagen, in the case of mutations in PPIB [49,51,52]. Consequently, collagen folding is delayed [49,[52][53][54] and a change in fibril assembly, crosslinking, and bone mineralization occurs [51,52,54]. The newly formed hydroxylysine residues are glycosylated by the addition of monosaccharides, such as galactose and glucose.…”

PrP-containing aggresomes are cytosolic components of an ER quality control mechanism

“…The analysis of PrP in the brain of CyPB KO mice CyPB KO mice (Cabral et al, 2014) and their wild-type siblings (strain C57BL/6, which were bred from Het X Het mating) were maintained under an NICHD-approved animal care program. Mice were euthanized at either 1 or 6 months of age, and brains were removed.…”

“…To examine this notion, we used cyclophilin-B-knockout mice (CyPB KO mice) (Cabral et al, 2014). Brains from six 6-month-old CyPB KO and of six age-matched wild-type siblings (three males and three females for each genotype) were homogenized and subjected to high-speed centrifugation to separate soluble PrP species from PrP aggregates.…”