ROLES OF P24 PROTEINS IN GLYCOSYLATION AND EXTRACELLULAR SIGNALING PATHWAYS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞表面和胞外蛋白上的复杂碳水化合物在细胞生长和分化的关键分子识别事件中发挥着重要作用。在人类中，新出现的先天性糖基化障碍(CDGs)包括与蛋白质糖基化异常有关的严重多系统综合征；大多数已识别的CDGs反映了糖生物合成途径的中断。最近的研究表明，糖基化异常和CDG的临床表现也可以由细胞内蛋白运输的缺陷引起。遗传上易驯化的简单生物为研究这些保守途径的调控机制提供了机会。我们已经在线虫中发现了三个基因，当它们突变时，会引起凝集素麦芽凝集素与角质层表面的结合，这表明它们在一个或多个糖基化途径的调节中发挥作用。这些突变使一个保守的蛋白质家族(P24)的成员失活，该家族与内质网对高尔基体运输的货物选择性有关。有趣的是，这些突变体显示出特定的神经元异常(我们的初步结果)，为探索p24活性在神经系统发育中的作用提供了机会。 我们在线虫中使用遗传和生化方法来实现以下目的：1)通过分析野生型和突变型胚胎细胞中代谢标记的糖蛋白和凝集素纯化组分，来检测p24活性与蛋白质糖基化状态的关系；2)利用双突变分析，探索作为p24活性下游靶点的细胞外信号通路；以及3)利用异常的凝集素与角质层表面的结合来寻找参与表面蛋白糖基化和运输的其他基因。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Complex carbohydrates on cell surface and extracellular proteins play essential roles in molecular recognition events critical to cell growth and differentiation. In humans, the emerging congenital disorders of glycosylation (CDGs) include severe multisystemic syndromes linked to abnormal protein glycosylation; most of the identified CDGs reflect disruptions of glycan biosynthetic pathways. Recent studies have revealed that glycosylation abnormalities and CDG clinical presentations can also be caused by defects in intracellular protein trafficking. Genetically tractable simple organisms provide an opportunity to study the regulatory mechanisms of these conserved pathways. We have identified three genes in the nematode Caenorhabditis elegans which, when mutated, elicit binding of the lectin Wheat Germ Agglutinin on the cuticle surface, suggesting a role in the modulation of one or more glycosylation pathways. These mutations inactivate members of a conserved protein family (p24) implicated in cargo selectivity of endoplasmic reticulum to Golgi transport. Interestingly, these mutants exhibit specific neuronal abnormalities (our preliminary results), offering the opportunity to explore the role of p24 activity on the development of the nervous system. We use genetic and biochemical approaches in C. elegans to pursue the following aims: 1) examine the relationship between p24 activity and protein glycosylation status, by analysis of metabolically labeled glycoproteins from wild type and mutant embryonic cells and lectin purified fractions; 2) explore extracellular signaling pathways as downstream targets of p24 activity, using double mutant analysis; and 3) exploit abnormal lectin binding to the cuticle surface to identify additional genes involved in surface protein glycosylation and trafficking.