Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease
Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease
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DOI：
10.1128/jb.187.13.4421-4429.2005
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发表时间：
2005-07-01
影响因子：
3.2
通讯作者：
Sokol, PA
中科院分区：
文献类型：
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作者：
Kooi, C;Corbett, CR;Sokol, PA
新绿脓杆菌ZmpA是典型的热溶酶样蛋白酶（如铜绿假单胞菌LasB和热溶蛋白芽孢杆菌）的前酶。zmpA基因采用pPRO-EXHTa His(6)标签表达系统表达，该系统在蛋白n端加入6 -His标签，重组zmpA采用ni -硝基三乙酸亲和层析纯化。重组His(6)-pre-pro-ZmpA （62 kDa）后，融合蛋白被自蛋白水解裂解为36-kDa（成熟的ZmpA）和27-kDa肽段。利用位点定向诱变来推断ZmpA活性位点残基的身份，并证实该酶进行了自蛋白水解裂解。用寡核苷酸诱变法将H-465替换为G（46）或A(465)，将E-377替换为A（377）或D-377，将H-380替换为P-380或A（380）。H-465、E-377或H-380的诱变导致了自催化活性和蛋白水解活性的丧失。H-380中两种取代的ZmpA均未检出。重组蛋白ZmpA的活性被EDTA和1,10菲罗啉抑制，表明它是一种锌金属蛋白酶。然而，ZmpA不受磷酰胺（一种典型的热溶酶样蛋白酶抑制剂）的抑制。折叠后的成熟ZmpA酶对多种底物具有蛋白水解活性，包括兽皮粉天蓝、IV型胶原、纤维连接蛋白、中性粒细胞α -1蛋白酶抑制剂、α(2)-巨球蛋白和γ干扰素，这表明cenocepacia B. ZmpA可能通过调节宿主免疫系统直接对宿主组织造成损伤或对宿主组织造成损伤。
Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H-465 with G(46), or A(465), E-377 with A(377) or D-377, or H-380 with P-380 or A(380). Mutagenesis of H-465, E-377, or H-380 resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H-380 was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.