THROMBOSPONDIN 4

凝血酶原蛋白是细胞外钙结合蛋白家族。 具有黏附和反黏附活性的蛋白质。血栓反应蛋白-1 是这个家庭中最具特点的成员。一直以来 被证明可以调节细胞的黏附、迁移和生长。的目标是 这项建议是为了表征凝血酶反应蛋白的两个新成员 吉恩家族。去神经后肌肉中凝血酶反应蛋白-4的上调 并支持神经元附着和轴突生长。软骨 寡聚基质蛋白(COMP或凝血酶反应蛋白-5)已被证明是 在骨质疏松症中含有突变。建议的研究是设计的 将从蛋白质获得的体外和体内数据相互关联 生物化学、细胞生物学和遗传学方法。具体重点将是 针对以下方面：(1)功能性质 凝血酶原蛋白-4和组氨酸。拟议研究的一个具体目的是 明确定位糖氨基多糖结合部位，钙 结合，以及通过产生这些蛋白质内的细胞结合 (1)保留功能性的蛋白水解性片段和融合蛋白 活性，(2)阻断功能的多克隆和单克隆抗体， (3)定点突变；(4)模拟或 抑制完整蛋白的活性。这些方法还将 用于定位支持神经突起的凝血酶反应蛋白-4的活性部位 外延生长。凝血酶敏感蛋白-4的正常和突变形式及其相互作用 将对域进行分析，以确定纠正以下异常的能力 凝血酶敏感蛋白-4缺陷小鼠和细胞系。(2)删除 凝血酶敏感蛋白-4基因。拟议研究的一个目标是删除 同源重组从小鼠基因组中分离出凝血酶敏感蛋白-4基因 在胚胎干细胞中。选定的胚胎干细胞将用于 产生嵌合体小鼠，可以培育出一种品系的小鼠 是杂合子或纯合子的凝血酶敏感蛋白-4缺乏症 两种遗传背景。如果纯合子不能存活，我们会 确定胚胎致死的发育阶段和原因。 如果纯合子动物存活，主要器官的形态 将对系统进行分析，特别关注大脑、心脏和 骨骼肌。其他成员获得赔偿的可能性 凝血酶反应蛋白基因家族将通过定量mrna进行研究。 水平，并通过与两个品系的小鼠杂交，这些小鼠缺乏其他 凝血酶原蛋白。(3)软骨发育不良的CoMP基因突变。一种特定的 拟议研究的目的是确定这些突变和其他突变是如何 导致PSACH和多发性骨骺发育不良(MED)。我们假设 这些软骨发育不良是由于 软骨细胞活性和生长过早停止或缺陷 软骨细胞与细胞外基质的相互作用。至 确定COMP突变在PSACH和MED中的作用，我们计划 检查突变对结构、生物合成和 COMP的功能突变蛋白将通过定点定位来制造 诱变和杆状病毒的表达。突变的蛋白质将是 与正常COMP相比，其(1)依赖于钙的折叠， (2)与伴侣的相互作用，(3)与糖胺聚糖结合的能力， (4)它们与细胞表面的相互作用。

The thrombospondins are a family of extracellular, calcium-binding proteins with adhesive and counter-adhesive activity. Thrombospondin-1 is the most extensively characterized member of the family. It has been shown to modulate cell adhesion, migration and growth. The objective of this proposal is to characterize two new members of the thrombospondin gene family. Thrombospondin-4 is upregulated in muscle after denervation and supports neuronal attachment and neurite outgrowth. Cartilage oligomeric matrix protein (COMP or thrombospondin-5) has been shown to contain mutations in bone dysplasias. The proposed study is designed to correlate in vitro and in vivo data that is obtained from protein biochemical, cell biological and genetic approaches. Specific focus will be directed toward the following areas:(1) Functional properties of thrombospondin-4 and COMP. A specific aim of the proposed study is to specifically map the sites for glycoasminoglycan binding, calcium binding, and cell binding within these proteins through the production of (1) proteolytic fragments and fusion proteins that retain functional activity, (2) polyclonal and monoclonal antibodies that block function, (3) site-directed mutagenesis, and (4) synthetic peptides that mimic or inhibit the activity of the intact proteins. These approaches will also be used to map active sites within thrombospondin-4 that support neurite outgrowth. The normal and mutant forms of thrombospondin-4 and its domains will be assayed for the ability to correct abnormalities in thrombospondin-4-deficient mice and cell lines. (2) Deletion of the thrombospondin-4 gene. A goal of the proposed study is to delete the thrombospondin-4 gene from the mouse genome by homologous recombination in embryonic stem cells. Selected embryonic stem cells will be used to produce chimeric mice that can be bred to establish a strain of mice that are heterozygous or homozygous for thrombospondin-4 deficiency in two genetic backgrounds. If the homozygotes are not viable, we will determine the developmental stage and cause of the embryonic lethality. If the homozygous animals are viable, the morphology of the major organ systems will be analyzed, with particular focus on brain, heart and skeletal muscle. The possibility of compensation by other members of the thrombospondin gene family will be investigated by quantitating mRNA levels and by crossing two strains of mice that are deficient in other thrombospondins. (3) COMP mutations in chondrodysplasias. A specific aim of the proposed study is to determine how these mutations and others cause PSACH and multiple epiphyseal dysplasia (MED). We hypothesize that these chondrodysplasias are a result of either decreased chondrocyte viability and premature cessation of growth or a defect in the interaction of chondrocytes with the extracellular matrix. To determine the role of COMP mutations in PSACH and MED, we plan to examine the effect of the mutations on the structure, biosynthesis and function of COMP. Mutant proteins will be made by site directed mutagenesis and baculovirus expression. The mutant proteins will be compared to normal COMP in terms of their (1) calcium-dependent folding, (2) interaction with chaperones, (3) ability to bind glycosaminoglycans, and (4) their interaction with cell surfaces.