Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease

Abstract

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(465) 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.

FIG. 1.

Solubilization and purification of preproZmpA by Ni-NTA chromatography. A. SDS-12.5% PAGE analysis of cell extracts of DH5α (pCK10) expressing preproZmpA. Lane 1, uninduced; lanes 2 to 5, induced with 0.6 mM IPTG for various times; lane 2, 1 h; lane 3, 2 h; lane 4, 3 h; lane 5, 4 h. The solid arrow indicates preproZmpA. B. SDS-12.5% PAGE of preproZmpA solubilized from inclusion bodies and purified using Ni-NTA chromatography. Lane 1, inclusion bodies solubilized in 6 M guanidine-HCl; lane 2, crude cell extract induced with 0.6 mM IPTG for 3 h; lanes 3 to 5, first three fractions of preproZmpA eluted from the Ni-NTA column with 100 mM imidazole.

FIG. 2.

Comparison of native, recombinant, and mutant forms of preproZmpA. A. Coomassie-stained SDS-12.5% PAGE gel. Lane 1, native ZmpA; lane 2, recombinant ZmpA; lane 3, ZmpA H465G; lane 4, ZmpA E377A; lane 5, ZmpA H380P; lane 6, ZmpA H465A; lane 7, ZmpA E377D; lane 8, ZmpA H380A; and lane 9, recombinant ZmpA. B. Western blot reacted with MAb 36-6-6 to native ZmpA. Lane 1, native ZmpA; lane 2, recombinant preproZmpA; and lane 3, ZmpA H465G. C. Western blot reacted with anti-His tag antibody. Lane 1, native ZmpA; lane 2, recombinant ZmpA; lane 3, ZmpA H465G; lane 4, ZmpA E377A; and lane 5, ZmpA H380P. The solid arrows indicate preproZmpA, and the open arrows indicate ZmpA.

FIG. 3.

Expression of wild-type and mutant zmpA proteins in K56-2-9. Western immunblot of whole cell lysates reacted with anti-ZmpA antibody. Lane 1, K56-2 (pUCP26); lane 2, K56-2-9 (pUCP26); lanes 3 to 9, K56-2-9 with wild-type or mutant zmpA in trans; lane 3, ZmpA; lane 4, ZmpA H465G; lane 5, ZmpA H465A; lane 6, ZmpA E377A; lane 7, ZmpAE377D; lane 8, ZmpA H380P; lane 9, ZmpAH380A; lane 10, native ZmpA. The solid arrow indicates preproZmpA and the open arrow indicates ZmpA.

FIG. 4.

Ability of recombinant ZmpA and native ZmpA to cleave biologically important substrates. Substrates were digested with native ZmpA (N), recombinant ZmpA (R), or no protease (−) for 24 to 48 h. Controls included native ZmpA only (n) and recombinant ZmpA (r) only digestions. Samples were separated by SDS-PAGE and stained with Coomassie blue. A. SDS-12.5% PAGE of α-1 proteinase inhibitor. B. SDS-7.5% PAGE of α₂-macroglobulin. C. SDS-7.5% PAGE of collagen (lanes 1 to 5) and fibronectin (lanes 6 to 10). D. Tricine 16%-PAGE of IFN-γ. E. SDS-12.5% PAGE of lactoferrin (lanes 1 to 3) and transferrin (lanes 4 to 6). F. SDS-12.5% PAGE of IgA (lanes 1 to 3), IgG (lanes 4 to 6), and IgM (lanes 7 to 9).