ANTHRAX TOXIN PROTECTIVE ANTIGEN IS ACTIVATED BY A CELL-SURFACE PROTEASE WITH THE SEQUENCE SPECIFICITY AND CATALYTIC PROPERTIES OF FURIN
ANTHRAX TOXIN PROTECTIVE ANTIGEN IS ACTIVATED BY A CELL-SURFACE PROTEASE WITH THE SEQUENCE SPECIFICITY AND CATALYTIC PROPERTIES OF FURIN
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DOI：
10.1073/pnas.89.21.10277
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发表时间：
1992-11-01
影响因子：
11.1
通讯作者：
LEPPLA, SH
中科院分区：
文献类型：
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作者：
KLIMPEL, KR;MOLLOY, SS;LEPPLA, SH
炭疽毒素的保护性抗原（PA）蛋白残基164-167的蛋白水解裂解是毒性活性的必要条件。细胞蛋白酶在这个序列上的切割，通常是在PA与细胞表面受体结合后进行的。我们试图通过确定其序列特异性和催化性质来鉴定这种蛋白酶。采用半随机盒式诱变技术，用Arg、Lys、Ser或Asn替换残基164-167产生突变体。对19个突变蛋白的分析表明，致死因子依赖性毒性需要Arg-Xaa-Xaa-Arg序列。在此基础上，又构建了3个突变体，序列分别为Ala-Lys-Lys-Arg、Arg-Lys-Lys-Ala和Arg-Ala-Ala-Arg。在这些突变蛋白中，Arg-Ala-Ala-Arg是有毒的，这证实了细胞蛋白酶可以识别序列Arg-Xaa-Xaa-Arg。含有Ala-Lys-Lys-Arg序列的突变体也有毒性，但需要比原来多13倍的蛋白质才能产生相同的毒性。这种序列特异性与普遍存在的枯草杆菌样蛋白酶furin相似，furin参与某些受体和生长因子前体的加工。因此，我们测试了重组可溶性furin是否能裂解PA。该呋喃衍生物能有效地裂解天然PA和Arg-Ala-Ala-Arg突变体，但不能裂解无毒PA突变体。此外，先前鉴定的furin抑制剂阻断了受体结合PA的裂解。这些数据表明，furin是激活PA的细胞蛋白酶，几乎所有类型的细胞表面都含有至少少量的furin。
Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required >13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the non-toxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.