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, 7 (28), 43088-43094

Importance of the Residue 190 on Bactericidal Activity of the Bactericidal/Permeability-Increasing Protein 5

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Importance of the Residue 190 on Bactericidal Activity of the Bactericidal/Permeability-Increasing Protein 5

Hanwei Wu et al. Oncotarget.

Abstract

The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5.

Keywords: BPI; charged residue; mutant; structural model.

Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1. Production and characterization of mBPI5 and Asp190Ala mutant
Lane M: markers; lane 1 and 5: whole cell lysates of mBPI5 and Asp190Ala mutant before IPTG induction; lane 2 and 6: sediment of mBPI5 and Asp190Ala mutant before IPTG induction; lane 3 and 7: sediment of mBPI5 and Asp190Ala mutant after IPTG induction; lanes 4 and 8: purified mBPI5 and Asp190Ala mutant.
Figure 2
Figure 2. Comparison of bactericidal activity of mBPI5 and its Asp190Ala mutant toward E.coli J5 and P.aeruginosa.
Both mBPI5 and Asp190Ala mutant can suppress E. coli J5 and P.aeruginosa in a concentration-dependent manner. Under relatively low concentrations (< 10 nM), the antibacterial activity of Asp190Ala mutant against two Gram-negative isolates is similar to that of wild-type. However, Asp190Ala mutant has a stronger effect on suppressing Gram-negative bacteria growth at higher concentrations. All data are shown as mean ± SD (*p < 0.05; **p < 0.001); bar SD.
Figure 3
Figure 3. Comparison of the binding affinity of mBPI5 and Asp190Ala mutant to lipids
(A) Asp190Ala mutant almost exists in the form of bound with lipid, whereas native mBPI5 binds weakly to liposomes. (B) The ratio of the amount of Asp190Ala mutant binding to lipid to the amount of it in supernatant increases about 8 folds compared with that of mBPI5. All data are shown as mean ± SD (*p < 0.05; **p < 0.001); bar SD.
Figure 4
Figure 4. The overall structure of mBPI5
(A) The structure of mBPI5 constructed by I-TASSER. mBPI5 shows a boomerang-shaped molecule formed by two domains, N-terminal and C-terminal domain. (B) Surface representation of mBPI5 according to the electrostatic character of residues. The negatively charged carboxylate of Asp 190 forms salt linkages with the positively charged guanidinium at the end of the side chain of arginine 188. Six positively charged residues, including Lys96, Lys102, Lys103, Lys187, Arg188 and Lys199, fold into the proximity of Asp 190.

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