Glycan alterations in C.elegans surface mutants

描述（由申请人提供）：后生动物的发展需要多种细胞类型的产生和复杂的多细胞结构的形态发生。遗传学研究已经确定了糖基化和蛋白聚糖生物合成在发育信号传导中的作用。秀丽隐杆线虫（Caenorhabditis elegans）是研究糖缀合物在多细胞生物中的作用的一个很好的模型系统，也是寄生线虫的一个原型模型，因为它具有完整的基因组序列和发育知识。在这个R2 I（探索性/发展性）资助申请中，我们希望探索糖缀合物表达与秀丽隐杆线虫发育事件之间的一种新的潜在功能联系。Srf-9、srf-8和srf-4（表面）是单位点突变体，具有多种缺陷，包括运动不协调、外阴突出、卵生异常、交配囊和性腺形态缺陷，但它们的表面异位结合了一种与n -乙酰氨基葡萄糖结合的凝集素，因此被分离出来。srf-9、srf-8和srf-4的突变与LIN- 12基因的突变相互作用，LIN- 12基因是LIN- 12 /NOTCH受体蛋白家族的成员，在发育过程中介导细胞间相互作用以指定细胞命运。我们将验证srf-9、srf-8和srf-4基因参与糖缀合物的生物合成、翻译后加工或分泌，从而将发育事件与糖缀合物表达联系起来的假设。具体而言，本研究将：1)通过角质层聚糖组分的结构表征，确定srf-9、srf-8和srf-4突变体中受影响的糖缀合物；2)结合单核苷酸多态性（SNP）定位和标记拯救实验，通过分子克隆鉴定srf-9、srf-8和srf-4基因。从这些研究中获得的信息将为研究这些基因在线虫发育事件中的作用提供工具。由于复杂表型是几种人类遗传疾病的标志，包括一些影响糖基化的疾病，因此拟议的研究将建立使用秀丽隐杆线虫srf突变体作为人类糖基化疾病模型的可行性。

DESCRIPTION (provided by applicant): Development of metazoans requires the production of multiple cell types and the morphogenesis of complex multicellular structures. Genetic studies have established roles for glycosylation and proteoglycan biosynthesis in developmental signaling. The nematode Caenorhabditis elegans is a good model system to study the role of glycoconjugates in multicellular organisms as well as a prototypic model for parasitic nematodes because of the knowledge of its complete genomic sequence and development. In this R2 I (Exploratory / Developmental) grant application, we wish to explore a novel potential functional linkage between expression of glycoconjugates and developmental events in C. elegans. Srf-9, srf-8 and srf-4 (surface) are single loci mutants that have multiple defects, including uncoordinated movement, protruding vulva, abnormal egg laying, and defective copulatory bursae and gonad morphology, yet they were isolated based on their ectopic surface binding to a lectin which binds to N-acetylglucosamine. Mutations in srf-9, srf-8 and srf-4 interact with mutations in the lin-12 gene, which a member of the LIN- 1 2/NOTCH family of receptor proteins that mediate cell-cell interactions to specify cell fate during development. We will test the hypothesis that the srf-9, srf-8 and srf-4 genes are involved in the biosynthesis, post-translational processing or secretion of glycoconjugates, and therefore link developmental events with glycoconjugates expression. Specifically, the proposed work will: 1) Define the glycoconjugates affected in srf-9, srf-8 and srf-4 mutants, through the structural characterization of cuticle glycan fractions, 2) Identify the srf-9, srf-8 and srf-4 genes through molecular cloning using a combination of Single Nucleotide Polymorphism (SNP) mapping, and marker rescue experiments. The information derived from these studies will provide the tools to study the role of these genes in developmental events in the nematode C. elegans. Because complex phenotypes are the hallmark of several human genetic disorders, including some that affect glycosylation, the proposed studies will establish the feasibility of using C. elegans srf mutants as a model for glycosylation disorders in humans.