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Molecular characterization of cell wall degrading proteases in Chlamydomonas reinhardtii

莱茵衣藻细胞壁降解蛋白酶的分子特征

基本信息

批准号：
63540533
负责人：
MATSUDA Yoshihiro
金额：
$ 1.15万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

In Chlamydomonas reinhardtii. the release or shedding of cell walls is observed in at least two different stages of the life cycle. First, zoospores, the products of mitosis in the vegetative cell cycle, hatch by breaking of their enclosing sporangial walls with hatching enzyme (HE). Second, gametes of two mating-types dissolve and shed their walls with lytic enzyme (LE) during mating as a necessary prelude to cell fusion. We have previously purified HE and LE and characterized then as a semine and metalloprotease, respectively. In the present work. we have further characterized these two cell wall degrading proteases with respect to their amino-terminal sequences and their cleavage specificities on peptides. We also found a protease inhibitor of RE, which occurred specifically during mitotic cell divisions. The results obtained are as follows:1. The N-terminal amino acid sequence of LE was EIYAGKPIDLRTIVYINDFS. This sequence was the same for LE which exists in vegetative cells, in gametic plus and minus cells and in mating medium after excretion. By this information of amino acid sequence, we have currently tried and succeeded in isolating cDNA clone of LE.2. By using a variety of synthetic model peptides. we found that LE cleaves the peptides bonds between two consecutive hydrophobic amino acids, whereas HE hydrodlyzes peptides at the carboxyl site of Lys or Arg.3. We were unable to find any HE activities in mature sporangia just before zoospore liberation. Instead, we found a specific protease inhibitor for HE in the cell homogenates. Partially purified inhibitor had a molecular mass of about 55 kDa and formed a revesible complex with HE. This inhibitor occurred only during mitotic cell divisions in the vegetative cell cycle and was accumulated in a space between the dividing cells and mother (sporangial) walls.

在莱茵衣藻中。细胞壁的释放或脱落至少在生命周期的两个不同阶段观察到。首先，游动孢子，营养细胞周期中有丝分裂的产物，通过孵化酶(HE)打破其包围的孢子囊壁而孵化。其次，两种交配型的配子在交配过程中用裂解酶(LE)溶解并脱壁，这是细胞融合的必要前奏。我们先前已经纯化了HE和LE，并分别将其鉴定为精氨酸和金属蛋白酶。在目前的工作中。我们进一步研究了这两种细胞壁降解酶的氨基末端序列及其在多肽上的切割特性。我们还发现了RE的一种蛋白水解酶抑制物，它发生在有丝分裂的细胞分裂过程中。结果如下：1.LE的N-端氨基酸序列为EIYAGKPIDLRTIVYINDFS。这一序列与存在于营养细胞、配子正负细胞和交配后排泄的交配介质中的LE相同。利用这一氨基酸序列信息，我们目前已经尝试并成功地分离了LE2的cDNA克隆。通过使用各种合成的模型多肽。我们发现，LE断裂了两个连续疏水氨基酸之间的多肽键，而HE则在Lys或Arg的羧基位置水解肽。在游动孢子解放前，我们没有在成熟的孢子囊中发现任何HE活性。取而代之的是，我们在细胞匀浆中发现了一种针对HE的特异性蛋白酶抑制剂。部分纯化的缓蚀剂相对分子质量约为55 kDa，并与HE形成显性络合物。这种抑制物只发生在营养细胞周期的有丝分裂细胞分裂期间，并积累在分裂细胞和母细胞(孢子囊)壁之间的空间。