Role of the Leprecan genes in skeletal formation

描述（由申请人提供）：我们对编码软骨相关蛋白的CRTAP基因的研究，以及发现其突变导致隐性成骨不全（OI）的发现，导致了对这种疾病负责的其他非胶原基因突变的鉴定。这些突变中的大多数发生在rER驻留蛋白中，其参与纤维胶原的修饰、折叠和组装。重要的是，虽然其中一些 其他方法，如脯氨酰3-羟基化，仍然相当未开发，值得更多的关注。由于脯氨酰3-羟基化存在于各种类型的胶原蛋白中，包括纤维状和非纤维状，因此鉴定负责这种修饰的分子组分以及它们如何在不同组织中一起工作将提供关于组织特异性基质分子产生和疾病新机制的重要线索。基于先前的脯氨酰3-羟基化复合物的表征，含有Crtap，P3 h1和CypB，我们现在建议确定Sc 65的作用，Sc 65是Leprecan基因家族的新成员，也是与Crtap最密切相关的蛋白质。我们的初步数据表明，Sc 65是一种rER蛋白，在骨骼中高度表达，突变时会导致低骨量表型。我们假设Sc 65介导与其他脯氨酰3-羟化酶，P3 h2和/或P3 h3的相互作用，有助于胶原蛋白的翻译后修饰的网站是不同于那些修改的Crtap复合物，突变时，导致OI的发展。为了验证这些假设，提出了以下具体目标：1）使用微CT、骨组织形态计量学、胶原质谱和FTIRI全面表征在Sc 65基因中插入纯合基因陷阱的小鼠的骨骼表型; 2）使用原代颅骨成骨细胞和离体骨细胞测定Sc 65突变对骨细胞分化的影响。来自Sc 65突变小鼠和WT对照小鼠的骨髓成骨和破骨细胞生成培养物; 3）确定Sc 65蛋白的亚细胞定位并通过免疫共沉淀和蛋白测序鉴定其相互作用配偶体;和4）通过产生Sc 65 floxed等位基因产生条件性敲除小鼠，将其固定在骨中并表征其骨骼表型。这项工作的成功完成将1）阐明Sc 65在骨骼发育和骨稳态中的新作用，2）鉴定Sc 65蛋白相互作用物，这将提供对胶原蛋白脯氨酰3-羟基化所涉及的分子机制的深入了解，3）增强对骨骼发育和其他组织中的Leprecan基因的理解，和4）提供了对这些非胶原蛋白在人类结缔组织疾病（包括OI）的病因学中的作用的进一步了解。 公共卫生相关性：Leprecan基因在骨骼形成中的作用Leprecan基因家族，包括Crtap、P3 h1、P3 h2、P3 h3和Sc 65，最近已被鉴定，并且其一些成员（Crtap和P3 h1）的突变与称为成骨障碍（OI）的人类疾病形式相关。在Leprecans中，Sc 65可能是特征最少的，尽管它与Crtap具有最高的相似性，并且可能代表paradox基因。我们的初步数据表明，它编码一种ER蛋白，并且与Crtap一样，它在骨形成过程中具有重要功能。我们将研究其在骨骼形成过程中的作用，这样做将获得更多的见解Crtap和胶原蛋白脯氨酰3-羟基化的功能。

DESCRIPTION (provided by applicant): Our studies of the CRTAP gene, encoding cartilage associated protein, and the discovery that its mutations cause recessive osteogenesis imperfecta (OI) have led to the identification of additional non-collagen gene mutations responsible for this disease. Most of these mutations occur in rER resident proteins that are involved in fibrillar collagen modification, folding and assembly. Importantly, while some of these processes are relatively well understood others, such as prolyl 3-hydroxylation, have remained quite unexplored and deserve more attention. Because prolyl 3-hydroxylation is found in various types of collagens, both fibrillar and non- fibrillar, the identification of the molecular componens responsible for this modification and how they work together in different tissues will provide important clues about tissue-specific matrix molecules production and novel mechanisms of disease. Based on previous characterization of the prolyl 3-hydroxylation complex, containing Crtap, P3h1 and CypB, we now propose to determine the role of Sc65, a novel member of the Leprecan gene family and the most closely related protein to Crtap. Our preliminary data show that Sc65 is a rER protein, highly expressed in the skeleton and when mutated causes a low bone mass phenotype. We hypothesize that Sc65 mediates interactions with the other prolyl 3-hydroxylase enzymes, P3h2 and/or P3h3, contributes to collagen post-translational modification at sites that are distinct from those modified by the Crtap complex and, when mutated, causes the development of OI. To test these hypotheses, the following specific aims are proposed: 1) Comprehensively characterize the skeletal phenotype of mice with a homozygous gene- trap insertion in the Sc65 gene using micro-CT, bone histomorphometry, collagen mass spectrometry and FTIRI; 2) Determine effects of Sc65 mutation on bone cell differentiation using primary calvarial osteoblasts and ex-vivo bone-marrow osteogenic and osteoclastogenic cultures from Sc65 mutant and WT control mice; 3) Determine the sub-cellular localization of the Sc65 protein and identify its interacting partners by co- immunoprecipitation and protein sequencing; and 4) Generate a conditional knock-out mouse by creation of a Sc65 floxed allele, inactivate it in bone and characterize its skeletal phenotype. Successful completion of this work will 1) elucidate a novel role for Sc65 in skeletal development and bone homeostasis, 2) identify Sc65 protein interactors which will provide insight into the molecular mechanisms involved in collagen prolyl 3- hydroxylation, 3) enhance understanding of the Leprecan genes in skeletal development and other tissues, and 4) provide further insight into the role of these non-collagenous proteins in the etiology of human connective tissue disease, including OI. PUBLIC HEALTH RELEVANCE: Role of Leprecan genes in skeletal formation The Leprecan family of genes, including Crtap, P3h1, P3h2, P3h3 and Sc65, has been recently identified and mutations in some of its members (Crtap and P3h1) are associated with forms of the human disease known as osteogenesis imperfecta (OI). Among Leprecans, Sc65 is probably the least characterized although it bears the highest similarity to Crtap and likely represents a paralog gene. Our preliminary data indicate that it encodes an ER protein and that, like Crtap, it has an important function during bone formation. We will investigate its role during skeletal formation and doing so will gain additional insights into the function of Crtap and of collagen prolyl 3-hydroxylation.