Sequence analysis and regulation of a gene encoding a cuticle-degrading serine protease from the nematophagous fungus Arthrobotrys oligospora
Sequence analysis and regulation of a gene encoding a cuticle-degrading serine protease from the nematophagous fungus Arthrobotrys oligospora
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DOI：
10.1099/13500872-142-7-1605
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发表时间：
1996-07-01
期刊：
影响因子：
2.8
通讯作者：
Tunlid, A
中科院分区：
文献类型：
--
作者：
Ahman, J;Ek, B;Tunlid, A
捕捉线虫的真菌少孢节杆菌产一种胞外丝氨酸蛋白酶(指定为PII)，在生物测定中固定自由生活的线虫，并分解线虫角质层的蛋白质。从PII中分离多肽，并对其进行部分测序。三个内部肽序列被用来设计合成的寡核苷酸，这使得随后可以从基因组库中分离编码PII的基因。推导的氨基酸序列表明，PII是以酶前原的形式合成的，它含有成熟的酶、信号序列和前肽，在酶分泌到培养基中之前被去除。PII的一级序列与属于枯草杆菌蛋白家族的子囊菌的其他几种丝氨酸蛋白酶具有高度的相似性。Northern分析表明，当真菌缺乏氮和碳时，PII可以表达，并且在包括线虫角质层碎片在内的各种可溶性和不溶性蛋白质的培养基中显著刺激了PII的表达。在更易代谢的氮(包括氨、硝酸盐和氨基酸)或葡萄糖的存在下，PII的mRNA水平和蛋白分解活性受到抑制。该酶的活性几乎完全被Phe-Val多肽和Phe氨基酸所抑制，而其mRNA水平却没有相应的降低。值得注意的是，已知具有相似结构的多肽由宿主(线虫)分泌，并刺激真菌感染结构(陷阱)的产生。
The nematode trapping fungus Arthrobotrys oligospora produces an extracellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticle. Peptides were isolated from PII and partly sequenced. Three internal peptide sequences were used to design synthetic oligonucleotides, which allowed the subsequent isolation of the gene encoding PII from a genomic library. The deduced amino acid sequence indicated that PII is synthesized as a preproenzyme containing the mature enzyme, a signal sequence and a propeptide that are removed before the enzyme is secreted into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belonging to the subtilisin family. Northern analysis demonstrated that PII was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to the medium of various soluble and insoluble proteins, including fragments of nematode cuticle. The levels of the mRNA as well as the proteolytic activity of PII were repressed in the presence of more easily metabolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibited by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar structures are known to be secreted by the host (nematode) and to stimulate the production of infection structures (traps) of the fungus.