Molecular characterization of cell wall degrading proteases in Chlamydomonas reinhardtii

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被观察到。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）打破其包裹的孢子囊壁而孵化。其次，两种交配类型的配子在交配过程中溶解并脱落其细胞壁，这是细胞融合的必要前奏。我们之前纯化了HE和LE，并分别将其表征为semine和metalloprotease。在目前的工作中。我们进一步表征了这两种细胞壁降解蛋白酶的氨基末端序列和它们在肽上的切割特异性。我们还发现了RE的蛋白酶抑制剂，这种抑制剂在有丝分裂细胞分裂时特异性地发生。得到的结果如下：1。LE的n端氨基酸序列为EIYAGKPIDLRTIVYINDFS。存在于营养细胞、配子正、负细胞和排泄后的交配培养基中的LE，其序列相同。利用这些氨基酸序列信息，我们目前已经尝试并成功地分离出了le2的cDNA克隆。利用多种模型肽合成。我们发现LE在两个连续的疏水氨基酸之间切割肽键，而HE在赖氨酸或赖氨酸3的羧基位点水解肽。在游动孢子释放前的成熟孢子囊中未发现HE活性。相反，我们在细胞匀浆中发现了HE的特异性蛋白酶抑制剂。部分纯化的抑制剂分子量约为55 kDa，与HE形成可逆络合物。这种抑制剂只发生在营养细胞周期的有丝分裂细胞分裂期间，并积聚在分裂细胞和母细胞（孢子囊）壁之间的空间中。

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.