Role of the Leprecan Genes in Skeletal Formation

描述(申请人提供)：我们对编码软骨相关蛋白的CRTAP基因的研究，以及它的突变导致隐性成骨不全(OI)的发现，导致了与这种疾病相关的其他非胶原基因突变的发现。这些突变大多发生在RER驻留蛋白中，这些蛋白参与胶原纤维的修饰、折叠和组装。重要的是，虽然其中一些 其他的过程，如3-羟基化，还没有被研究过，应该得到更多的关注。由于Prolyl 3-羟化存在于各种类型的胶原中，无论是纤维的还是非纤维的，识别负责这种修饰的分子成分以及它们如何在不同的组织中协同工作将为组织特异性基质分子的产生和新的疾病机制提供重要线索。基于先前对含有CrTAP、P3h1和CypB的Pro-3-羟基化复合体的鉴定，我们现在建议确定Sc65的作用，Sc65是Leprecan基因家族中的一个新成员，也是与CrTAP关系最密切的蛋白质。我们的初步数据显示，Sc65是一种RER蛋白，在骨骼中高表达，当突变时会导致低骨量表型。我们推测，Sc65介导与其他Pro 3-羟基酶P3h2和/或P3h3的相互作用，在不同于CRTAP复合体修饰的位置促进胶原翻译后修饰，当突变时，导致OI的发生。为了验证这些假说，提出了以下具体目标：1)利用显微CT、骨组织形态计量学、胶原质谱仪和FTIRI技术综合表征Sc65基因纯合子插入的小鼠的骨骼表型；2)通过原代颅骨成骨细胞和体外培养的Sc65突变小鼠和WT对照小鼠的骨髓成骨和破骨细胞培养，确定Sc65突变对骨细胞分化的影响；3)通过免疫共沉淀和蛋白质测序确定Sc65蛋白的亚细胞定位并确定其相互作用伙伴；4)建立Sc65等位基因的条件性敲除小鼠，使其在骨骼中失活，并对其骨骼表型进行鉴定。这项工作的成功完成将1)阐明Sc65在骨骼发育和骨骼动态平衡中的新角色，2)确定Sc65蛋白的相互作用，这将有助于深入了解胶原Pro-3-羟化所涉及的分子机制，3)增强对Leprecan基因在骨骼发育和其他组织中的理解，以及4)进一步深入了解这些非胶原蛋白在包括OI在内的人类结缔组织疾病的病因学中的作用。

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.