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Studies on Differentiation Mechanism of Dictyostelium discoideum

盘基网柄菌分化机制的研究

基本信息

批准号：
12680642
负责人：
FUJII Shigeru
金额：
$ 2.3万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680642/
关键词：
Dictyostelium differentiation factor glycoprotein protein structure sugar chains 細胞性粘菌

项目摘要

In the previous study, we have purified a factor inducing prespore cell differentiation with an apparent molecular mass of 106 kDa on SDS-PAGE, which we named psi-factor (prespore-cell-inducing factor). We analysed four partial amino acid sequences of the purified psi-factor, and synthesized an oligonucleotide corresponding to one partial amino acid sequence. By using it as a probe, we isolated cDNA clones encoding psi-factor. They contained the full-length psi-factor open reading frame with 1674 mucleotides, which encoded a protein of 557 amino acid residues. The amino acid sequence shows that the factor is a novel protein containing both proline- and cysteine- rich regions. The analysis of the N-terminal sequence of the purified psi-factor indicates that the first 19 amino acid residues are severed and the matured secreted psi-factor consists of 538 amino acid residues with a molecular mass of 60 kDa. This value is much smaller than the apparent molecular mass estimated by SDS-PAGE.. The difference may come from addition of sugar chains to the protein. The amino acid sequence shows six possible Asn-glycosylation sites, Asn-Xaa-Ser/Thr motifs. When psi-factor was treated with endoglycosidase H, its apparent molecular mass decreased on SDS-PAGE. It was strongly bound with ConA-agarose. These results indicated that psi-factor is a glycoprotein with Asn-linked carbohydrates. Knockout mutant Ax2 strains of the psi-factor gene were prepared by targeted integration. Rescuing the mutant strains by transforming actin 15-psi-factor expression vector caused overproduction of psi-factor protein and higher prespore-cell inducing activity. These results show that psi-factor is one of important differentiation factors in Dictyosteliium discoideum.

在前期的研究中，我们纯化了一个诱导前孢子细胞分化的因子，经SDS-PAGE分析，其表观分子量为106 kDa，我们将其命名为psi-factor（prespore-cell-inducing factor）。我们分析了纯化的PSI-因子的四个部分氨基酸序列，并合成了对应于一个部分氨基酸序列的寡核苷酸。通过使用它作为探针，我们分离了编码psi因子的cDNA克隆。它们含有全长psi因子开放阅读框，含1674个核苷酸，编码557个氨基酸残基的蛋白质。氨基酸序列表明，该因子是一种新的蛋白质，含有脯氨酸和半胱氨酸丰富的区域。纯化的PSI-因子的N-末端序列的分析表明，前19个氨基酸残基被切断，成熟的分泌型PSI-因子由538个氨基酸残基组成，分子量为60 kDa。该值远小于通过SDS-PAGE估计的表观分子量。这种差异可能来自于蛋白质中添加了糖链。氨基酸序列显示六个可能的Asn-糖基化位点，Asn-Xaa-Ser/Thr基序。当psi-因子与内切糖苷酶H处理时，其表观分子量在SDS-PAGE上降低。它与ConA-琼脂糖强烈结合。这些结果表明，psi因子是一种糖蛋白与天冬酰胺连接的碳水化合物。通过靶向整合制备psi因子基因的敲除突变体Ax 2菌株。通过转化actin 15-psi-因子表达载体拯救突变株，导致psi-因子蛋白过量产生和更高的前孢子细胞诱导活性。这些结果表明，PSI因子是盘基网柱藻分化的重要因子之一。