Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.
Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.
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DOI：
10.1371/journal.pgen.1004465
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发表时间：
2014-06
期刊：
影响因子：
4.5
通讯作者：
Marini JC
中科院分区：
文献类型：
--
作者：
Cabral WA;Perdivara I;Weis M;Terajima M;Blissett AR;Chang W;Perosky JE;Makareeva EN;Mertz EL;Leikin S;Tomer KB;Kozloff KM;Eyre DR;Yamauchi M;Marini JC
亲环蛋白B （Cyclophilin B, CyPB）由PPIB编码，是一种位于内质网的肽基脯氨酸顺式反式异构酶（PPIase），它独立起作用，是胶原脯氨酸3-羟基化复合物的一个组成部分。CyPB被认为是催化胶原折叠限速步骤的主要PPIase。PPIB突变导致隐性遗传成骨不全IX型，一种中重度至致死性骨发育不良。为了研究CyPB在胶原折叠和翻译后修饰中的作用，我们产生了再现OI表型的Ppib - / -小鼠。敲除（KO）小鼠体积小，股骨面骨矿物质密度（aBMD）、总容积骨量（BV/TV）和力学性能降低，股骨脆性增加。皮肤、成纤维细胞、股骨和颅骨成骨细胞中不存在Ppib转录本，western blots显示KO成骨细胞和成纤维细胞中不存在CyPB。在KO细胞和组织中仅检测到残留的（2-11%）胶原脯氨酸3-羟基化。在没有CyPB的情况下，胶原蛋白折叠更慢，支持其在折叠中的限速作用。然而，用环孢素A处理KO细胞会导致折叠进一步延迟，表明可能存在另一种胶原PPIase。我们证实并扩展了CyPB在支持胶原赖基羟化酶（LH1）活性中的作用。Ppib−/−成纤维细胞和成骨细胞胶原的总赖基羟基化正常，而胶原二糖基化增加。液相色谱/质谱（LC/MS）分析骨和成骨细胞I型胶原发现螺旋赖氨酸羟基化位点特异性改变，特别是螺旋交联残基K87羟基化显著降低。因此，低羟基形式的二价和三价交联在KO骨中显著增加，导致总交联增加，螺旋羟基赖氨酸与赖氨酸衍生的交联比率降低。交联模式的改变与培养基质中胶原沉积减少、组织中纤维结构改变和骨强度降低有关。这些研究证明了CyPB对胶原羟基化的间接调节作用的新结果，影响胶原糖基化，交联和纤维形成，这有助于维持骨力学性能。成骨不全症（Osteogenesis imperfecta， OI），或称脆性骨病，其特点是由于轻微创伤和生长缺陷而易发生骨折。胶原脯氨酸3-羟基化复合物成分CRTAP、P3H1和CyPB的缺乏分别导致隐性VII型、VIII型和IX型OI。我们之前已经表明，内质网内的相互保护解释了CRTAP和P3H1突变患者重叠的严重表型。然而，CyPB缺乏症患者的骨发育不良在表型和I型胶原生物化学方面是不同的。使用IX型OI敲除小鼠模型，我们已经证明CyPB是主要的，尽管不是唯一的，肽基脯氨酸顺式反式异构酶，催化胶原折叠的限速步骤。CyPB对于胶原蛋白脯氨酸3-羟基化复合物的活性也是必需的；在缺乏CyPB的情况下，胶原α1(I) P986修饰缺失。出乎意料的是，CyPB还以组织特异性、细胞特异性和残基特异性的方式影响胶原螺旋赖基羟基化。因此，CyPB直接促进胶原折叠，但也通过与内质网中其他胶原修饰酶的相互作用间接调节胶原羟基化、糖基化、交联和纤维形成。
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. Osteogenesis imperfecta (OI), or brittle bone disease, is characterized by susceptibility to fractures from minimal trauma and growth deficiency. Deficiency of components of the collagen prolyl 3-hydroxylation complex, CRTAP, P3H1 and CyPB, cause recessive types VII, VIII and IX OI, respectively. We have previously shown that mutual protection within the endoplasmic reticulum accounts for the overlapping severe phenotype of patients with CRTAP and P3H1 mutations. However, the bone dysplasia in patients with CyPB deficiency is distinct in terms of phenotype and type I collagen biochemistry. Using a knock-out mouse model of type IX OI, we have demonstrated that CyPB is the major, although not unique, peptidyl prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding. CyPB is also required for activity of the collagen prolyl 3-hydroxylation complex; collagen α1(I) P986 modification is lost in the absence of CyPB. Unexpectedly, CyPB was found to also influence collagen helical lysyl hydroxylation in a tissue-, cell- and residue-specific manner. Thus CyPB facilitates collagen folding directly, but also indirectly regulates collagen hydroxylation, glycosylation, crosslinking and fibrillogenesis through its interactions with other collagen modifying enzymes in the endoplasmic reticulum.