Structural and Functional Studies of Invertebrate Classical Cadherins

描述（由申请人提供）： 本文提出的研究目标是利用X射线晶体学结构测定和结构指导的功能实验来确定果蝇神经（DN）-钙粘蛋白（一种重要的细胞间粘附蛋白）功能的分子基础。DN-cadherin在果蝇视觉系统中起着重要的作用。在该项目的后期阶段，一旦确定了适当的DN-钙粘蛋白结构，我们将采用果蝇遗传分析的强大方法来探测DN-钙粘蛋白在体内的功能机制。果蝇基因组编码3种经典的钙粘蛋白，所有这些都在眼睛中表达。这些钙粘蛋白中只有两种在粘附中起作用，但只有DN-钙粘蛋白的粘附活性是感光细胞传入靶选择所需的。虽然多达5种不同的经典钙粘蛋白在脊椎动物中具有类似的功能，但12种不同的DN-钙粘蛋白亚型在果蝇中由选择性剪接编码，这表明DN-钙粘蛋白可能具有多种作用。已知脊椎动物经典钙粘蛋白通过充分表征的结合机制在其末端N-末端结合而在细胞间粘附中起作用。脊椎动物经典的钙粘蛋白分子在并列细胞上通过相互结合或交换其N末端β链形成粘附二聚体。这种链交换通过将来自一个配偶体钙粘蛋白的交换链的高度保守的色氨酸残基插入到来自并置细胞的配偶体分子中的保守疏水口袋中来锚定。虽然这种相互作用中的关键残基在脊椎动物物种中是高度保守的，但脊椎动物和无脊椎动物经典钙粘蛋白的比对表明，这些残基在无脊椎动物分子中是不保守的。这表明无脊椎动物钙粘蛋白，包括DN-钙粘蛋白，可能通过不同的机制在粘附中起作用。此外，脊椎动物和无脊椎动物的经典钙粘蛋白在其胞外结构域的总体数量和结构上是惊人不同的：成熟的脊椎动物经典钙粘蛋白含有5个胞外钙粘蛋白（EC）结构域（EC 1 -5），而DN-钙粘蛋白含有10至15个（取决于剪接形式），并且一些EC结构域似乎与脊椎动物EC结构域显著不同。与脊椎动物钙粘蛋白一样，DN-钙粘蛋白可能需要弗林蛋白酶样蛋白酶的蛋白水解加工以实现其成熟形式。 公共卫生相关性：钙粘蛋白功能和功能障碍的知识适用于人类健康的多个方面，因为钙粘蛋白在脊椎动物生物体中几乎所有实体组织的发育中发挥作用。腭裂41、皮肤病42和最终导致失明的青少年黄斑营养不良（HJMD）43可以由人类经典钙粘蛋白基因的突变引起。事实上，DN-钙粘蛋白的研究与视觉系统发育和疾病特别相关。

DESCRIPTION (provided by applicant): The goal of the research proposed here is to use structure determination by X-ray crystallography and structure-guided functional experiments to determine the molecular basis of function for Drosophila neural (DN)-cadherin, an important intercellular adhesion protein. DN-cadherin plays an important role in patterning the Drosophila visual system. At later stages in the project, once appropriate DN-cadherin structures have been determined, we will employ the powerful methods of Drosophila genetic analysis to probe the mechanism of DN-cadherin function in vivo. The Drosophila genome encodes 3 classical cadherins, all of which are expressed in the eye. Only two of these cadherins function in adhesion, but it is only the adhesive activity of DN-cadherin is required for photoreceptor afferent-target selection. Although up to 5 distinct classical cadherins have been implicated in analogous functions in vertebrates, 12 distinct DN-cadherin isoforms are encoded by alternative splicing in the fly, suggesting the possibility that DN-cadherin may subsume multiple roles. Vertebrate classical cadherins are known to function in intercellular adhesion through binding at their extreme N-termini through a well-characterized binding mechanism. Vertebrate classical cadherin molecules presented on juxtaposed cells form an adhesive dimer by the reciprocal binding, or "swapping", of their Nterminal (3-strands. This strand-exchange is anchored by the insertion of highly conserved tryptophan residues from the exchanged strand from one partner cadherin into a conserved hydrophobic pocket in the partner molecule from the apposed cell. While key residues in this interaction are highly conserved among vertebrate species, alignments of vertebrate and invertebrate classical cadherins show that these residues are not conserved in the invertebrate molecules. This suggests that invertebrate cadherins, including DN-cadherin, are likely to function in adhesion by distinct mechanisms. Moreover, vertebrate and invertebrate classical cadherins are strikingly different in the overall number and architecture of their extracellular domains: mature vertebrate classical cadherins contain five extracelluar cadherin (EC) domains (EC1-5), whereas DN-cadherin contains between 10 and 15 (depending on splice form), and some EC domains appear to be significantly diverged from vertebrate EC domains. Like vertebrate cadherins, DN-cadherin may require proteolytic processing by a furin-like protease to achieve its mature form. PUBLIC HEALTH RELEVANCE: Knowledge of cadherin function and dysfunction is applicable to multiple facets of human health because of the role played by cadherins in the development of almost all solid tissues in vertebrate organisms. Cleft palate41, skin disorders42, and juvenile macular dystrophy eventually leading to blindness (HJMD)43 can be caused by mutations in human classical cadherin genes. Indeed, the study of DN-cadherin is particularly relevant in visual system development and disease.