Growth Factor Interactions with Type II Procollagen

描述（由申请人提供）：关节软骨的形成 需要软骨生成细胞，生长因子， 和基质大分子，包括II型前胶原。软骨形成是 其特征在于产生II型的选择性剪接形式， 在氨基末端含有富含半胱氨酸（CR）结构域的前胶原（IIA） (NH2)前肽这个外显子2编码的结构域与BMP-2结合， 与其他结合BMP的分泌蛋白中的多个拷贝中发现的基序 (e.g.,和弦和sog）。这些骨形成蛋白结合蛋白在细胞凋亡中起关键作用。 类型确定和形态发生。我们将测试假设， IIA型前胶原中CR结构域的空间呈递对于 与生长因子的相互作用。IIA NH 2-前肽的研究已经完成， 这受到从组织中分离胶原蛋白前肽的复杂性的阻碍。我们， 因此，产生了由IIA NH 2-前肽组成的嵌合蛋白 连接至C 00 H-末端C-型凝集素三聚化盒以模拟 天然前胶原中IIA CR结构域的呈递。因此，我们将 使用该构建体及其突变体构建体有三个目的：我们将：1） 确定单独的外显子2结构域是否足以用于最佳BMP-2 结合或全长前肽是否是正确结合所必需的。 这些外显子2结构域的空间排列; 2）比较生长 不同的野生型和突变体IIA NH 2-前肽的因子结合活性 外显子2结构域的数量和分布;和3）检查功能 在体外的生物系统中。的结构鉴定 野生型前肽将使我们能够在CR中产生突变的蛋白质， 结构域来解释参与生长因子结合的重要氨基酸。的 这些分子的结构将使用生物化学和 免疫学技术，以及它们与BMP-2的体外相互作用， 使用结合分析进行研究。为了第三个目标，我们将使用一个高度 测量BMP依赖性延伸（分化）的可重复测定 的透镜上皮细胞的生物活性，以监测野生型或 含突变外显子2的前肽。拟议的研究将共同 提供了关于外显子2序列的功能作用的重要数据， 软骨发育和修复。获得的数据将有助于 制定战略，研究过度表达选定的 转基因小鼠中的突变体。

DESCRIPTION (provided by applicant): The formation of articular cartilage requires a complex interplay between cartilage producing cells, growth factors, and matrix macromolecules, including type II procollagen. Chondrogenesis is characterized by the production of an alternatively-spliced form of type II procollagen (IIA) containing a cysteine-rich (CR) domain in the amino-terminal (NH2) propeptide. This exon 2-encoded domain binds to BMP-2 and is homologous to motifs found in multiple copies in other secreted proteins that bind BMPs (e.g., chordin and sog). These BMP-binding proteins play critical roles in cell type determination and morphogenesis. We will test the hypothesis that the spatial presentation of CR domains in type IIA procollagen is critical for interactions with growth factors. Studies of the IIA NH2-propeptide have been hampered by the complexity of isolating collagen propeptides from tissues. We, therefore, produced a chimeric protein consisting of the IIA NH2-propeptide linked to a C00H-terminal C-type lectin trimerization cassette to mimic the presentation of the IIA CR domains in native procollagen. Accordingly, we will use this construct and mutant constructs thereof for three aims: We will: 1) determine whether the exon 2 domain alone is sufficient for optimal BMP-2 binding or whether the full-length propeptide is necessary for the correct spatial arrangement of these exon 2 domains; 2) compare the growth factor-binding activity of wild-type and mutant IIA NH2-propeptides that differ in the number and distribution of exon 2 domains; and 3) examine the function of these constructs in a biological system in vitro. Structural elucidation of wild-type propeptides will allow us to create proteins with mutations in the CR domain to decipher important amino acids involved in growth factor binding. The structure of these molecules will be characterized using biochemical and immunological techniques, and their in vitro interactions with BMP-2 will be studied using binding assays. For the third aim, we will use a highly reproducible assay that measures the BMP-dependent elongation (differentiation) of lens epithelial cells to monitor the biological activity of wild-type or mutant exon 2-containing propeptides. Together, the proposed studies will provide important data about the functional roles of exon 2 sequences in cartilage development and repair. The data obtained will assist with the development of strategies to examine the effect of over-expressing selected mutants in transgenic mice.