CLONING AND DISRUPTION OF THE GENE ENCODING AN EXTRACELLULAR METALLOPROTEASE OF ASPERGILLUS-FUMIGATUS
CLONING AND DISRUPTION OF THE GENE ENCODING AN EXTRACELLULAR METALLOPROTEASE OF ASPERGILLUS-FUMIGATUS
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DOI：
10.1111/j.1365-2958.1994.tb01327.x
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发表时间：
1994-12-01
影响因子：
3.6
通讯作者：
MONOD, M
中科院分区：
文献类型：
--
作者：
JATONOGAY, K;PARIS, S;MONOD, M
当真菌在胶原蛋白作为唯一氮源和碳源的存在下培养时，烟曲霉分泌丝氨酸碱性蛋白酶（ALP）和金属蛋白酶（MEP）。编码ALP的基因先前已被分离和表征。我们在这里报告的MEP编码基因的克隆和测序。从A.使用合成的寡核苷酸作为探针的烟曲霉文库。延伸的推导的氨基酸序列被认为是一致的N-末端氨基酸序列的MEP和内部肽序列。该酶的氨基酸序列含有一个推定的活性位点序列HEYTH，与其他细菌和真核生物的锌金属蛋白酶的活性位点同源。序列分析表明，MEP具有一个由245个氨基酸残基组成的前前区，前区的388个氨基酸残基（分子量为42 kDa）。构建了在体外中性pH下蛋白水解活性缺乏的alp mep突变体，并在小鼠模型中测试了其致病性。野生型菌株与alp-mep双突变株的致病性无明显差异，提示ALP和MEP不是A.烟熏。
Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases, Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.