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

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

Procollagen Triple Helix Assembly: An Unconventional Chaperone-Assisted Folding Paradigm

Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30–34°C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50–200 µM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization

Deficiency of Cartilage-Associated Protein in Recessive Lethal Osteogenesis Imperfecta

Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes

ER-like molecular crowding with nonspecific proteins does not affect procollagen thermal stability.

<p>A and B. Normalized DSC thermograms (A) and apparent T_m (B) at 1°C/min heating in PBS without and with 90 mg/ml BSA, 90 mg/ml IgG, or 100 mg/ml lysozyme (the thermograms in A and the corresponding bars in B have the same colors). C. Unfolding kinetics of procollagen at 37.5°C. In PBS with 90 mg/ml BSA, native procollagen fractions (inset, squares) were measured from the area under 1°C/min DSC thermograms of sample aliquots (blue tracings). In PBS without BSA, native procollagen fractions were measured from CD as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001029#pone-0001029-g001" target="_blank">Fig. 1B</a>.</p

Divalent ions do not affect thermal stability of procollagen.

<p>Normalized denaturation thermograms (A) and apparent T_m (B) measured by DSCD at 0.05°C/min scanning rate in PBS, DPBS (PBS with 1 mM CaCl₂ and 0.5 mM MgCl₂), TBS (50 mM Tris, 150 mM NaCl), and TBS with 10 mM CaCl₂. All buffers had neutral pH 7.1–7.5. The thermograms in A have the same colors as the corresponding bars in chart B.</p

Procollagen triple helix spontaneously refolds below but not above 34°C.

<p>A. Kinetics of 0.1 mg/ml procollagen refolding in PBS after 10 min denaturation of the triple helices at 45°C (monitored by CD as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001029#pone-0001029-g004" target="_blank">Fig. 4</a>). B and C. Native triple helix refolding in PBS without (B) and with 90 mg/ml BSA (C) after an initial DSC scan from 25 to 50°C at 1°C/min. The fraction of refolded native procollagen (insets) was measured from the area under the DSC thermograms (colored tracings) after overnight equilibration in the DSC instrument at indicated temperatures following the initial denaturation scan (native control). A second scan without the overnight equilibration is shown by the yellow line in C.</p

HSP47 may allow procollagen triple helix folding at normal and elevated body temperatures.

<p>The free energy of triple helix stabilization <i>δ</i>(Δ<i>G</i>) and the corresponding maximum triple helix folding temperature <i>T</i>₀+<i>δT</i> were calculated from Eqs. (5),(6) based on the HSP47 binding sites and dissociation constants reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001029#pone.0001029-Koide2" target="_blank">[40]</a>, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001029#s4" target="_blank">Methods</a> (Theoretical analysis). Two possible values of the dissociation constant <i>K_d</i>(OGxRG) = 4.6 µM (solid line) and <i>K_d</i>(OGxRG) = 0.94 µM (dashed line) were used to account for the uncertainty in HSP47 binding at OGxRG, where x is a variable amino acid.</p

Native structure of procollagen triple helix spontaneously refolds at 30°C after mild denaturation.

<p>A. Kinetics of triple helix recovery at 30°C monitored by CD at 223.8 nm in 0.1 mg/ml procollagen solution in PBS after 10 min denaturation at indicated temperatures. B. DSCD thermograms (0.05°C/min) of a native control sample and the refolded procollagen solutions after 10 hour equilibration. The native control has a single, narrow peak at ∼41°C. Additional peaks at lower temperature in the refolded samples originate from shorter, less stable, gelatin-like helices.</p