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

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

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development

Absence of <i>Ppib</i> expression affects bone development.

<p>(A) Staining of newborn skeletons with Alizarin red (bone) and Alcian blue (cartilage) reveals undermineralization of calvaria and ribs. Homozygous mice have smaller size of whole skeleton and long bones, and a deformed rib cage. (B) X-rays of 8 week-old mice. (C) DXA analysis of 8 week-old mice (n = 10/genotype).</p

Synthesis of type I collagen.

<p>(A) Western blots of cell lysates with antibodies to collagen 3-hydroxylation complex components. Lysates are derived from two independent cultures for each genotype. (B) SDS-Urea PAGE analysis of steady-state labeled type I collagen from wild-type (+/+), heterozygous (+/−) and homozygous (−/−) fibroblasts (FB) and osteoblasts (OB). (C) Differential scanning calorimetry (DSC) analysis reveals no differences in thermal stability (T_m) of type I collagen secreted by fibroblast cultures.</p

Post-translational modification of type I collagen lysine residues.

<p>*p<0.05 between +/+ and −/−;</p>#<p>p<0.05 between +/− and −/−;</p><p>no significant difference between +/+ and +/− in bone.</p

Whole bone structural and mechanical properties.

<p>(A) Structural parameters of wild-type (+/+) and homozygous (−/−) femora at 8 weeks of age characterize reduced bone formation in CyPB-deficient mice (n = 9/genotype). <i>Left</i>, 3D reconstructions illustrate reduced trabecular and cortical bone volumes. <i>Right</i>, Trabecular parameters are decreased in homozygous (−/−) femora, including reduced bone volume (Tb BV/TV, p = 0.01), thickness (Tb Th, p = 0.04) and number (Tb N, p = 0.01). Cortical bone parameters of CyPB-deficient mice are also reduced, with reductions in cortical thickness (Ct Th, p = 0.003) and area (Ct Ar, p = 0.02). (B) <i>Left</i>, Representative load-displacement curve demonstrating differences between samples selected for median post-yield displacement. <i>Right</i>, CyPB-deficient femora are weaker in yield (Yd Load, p = 1.9×10⁻⁶), ultimate (Ult Load, p = 8.9×10⁻⁷) and failure loads (p = 2.1×10⁻⁵), with reduced stiffness (p = 0.001). <i>Ppib</i>^−/− femora are also more brittle than wild-type femora, as demonstrated by decreased post-yield displacement (PYD, p = 0.001). Reduced toughness of <i>Ppib</i>^−/− femora is evident by decreases in the elastic and plastic energy (E) values (p = 0.001 and 0.0003, respectively).</p

Dysregulation of collagen deposition and fibril assembly.

<p>(A) <i>Left</i>, Deposition of type I collagen by osteoblasts into extracellular matrix in culture. Post-confluent cultures were pulsed for 24 hr, followed by serial extraction of incorporated collagens from the media (M), neutral salt (NS), acid soluble (AA, immaturely crosslinked) and pepsin soluble (P, maturely crosslinked) fractions of the matrix. <i>Right</i>, Matrix collagen to cell organics ratio from Raman micro-spectroscopy shows decreased collagen content in matrix deposited by homozygous <i>Ppib</i>-null (−/−) versus wild-type (+/+) osteoblasts in culture (p = 0.002). (B) Transmission electron micrographs of femoral and dermal collagen fibrils from 8 week-old wild-type (+/+) and CyPB-deficient mice (−/−). Diameters of 200 dermal fibrils were measured for each sample and plotted, right. (C) Quantitation of divalent crosslinks in murine humeri reveals increased HLNL (hydroxylysinonorleucine) crosslinks, which require helical lysine residues, but no change in DHLNL (dihydroxylysinonorleucine) crosslinks, which involve helical hydroxylysine residues. (D) Quantitation of trivalent crosslinks in murine humeri and femora. Total pyridinoline crosslinks are increased due to an increase in lysyl pyridinoline (LP), but not hydroxylysyl pyridinoline (HP) crosslinks in bone.</p