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Crystal structure of a charge engineered human lysozyme having enhanced bactericidal activity.

Gill A, Scanlon TC, Osipovitch DC, Madden DR, Griswold KE - PLoS ONE (2011)

Bottom Line: A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules.Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues.The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

ABSTRACT
Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

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Proteolytic Susceptibility Analysis of Lysozymes.An SDS-PAGE gel was Coomassie stained for total protein. Lane 1 = Kaleidoscope prestained molecular weight standards. Lanes 2–5 = 900 ng of the engineered double mutant. Lane 2 is an untreated control, and lanes 3, 4, and 5 are 50 µg/ml treatments with human neutrophil elastase, cathepsin G, and proteinase 3, respectively. Lanes 6–9 are the wild type hLYS arrayed in a similar fashion. Lanes 10–13 are bovine serum albumin (BSA) arrayed in a similar fashion. The wild type hLYS and engineered double mutant migrate just below the 15 kDa band as expected (lanes 2–9). The BSA control migrates between the 50 and 75 kDa bands as expected (lane 10). The three proteases can be seen migrating between the 25 and 37 kDa bands in the treatment lanes. Neither wild type hLYS nor the double mutant is degraded by the proteases, while BSA is fully degraded by all three. Similar results were obtained with both 5 µg/ml and 0.5 µg/ml protease treatments (data not shown), although BSA was only partially degraded by 0.5 µg/ml proteinase 3.
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pone-0016788-g005: Proteolytic Susceptibility Analysis of Lysozymes.An SDS-PAGE gel was Coomassie stained for total protein. Lane 1 = Kaleidoscope prestained molecular weight standards. Lanes 2–5 = 900 ng of the engineered double mutant. Lane 2 is an untreated control, and lanes 3, 4, and 5 are 50 µg/ml treatments with human neutrophil elastase, cathepsin G, and proteinase 3, respectively. Lanes 6–9 are the wild type hLYS arrayed in a similar fashion. Lanes 10–13 are bovine serum albumin (BSA) arrayed in a similar fashion. The wild type hLYS and engineered double mutant migrate just below the 15 kDa band as expected (lanes 2–9). The BSA control migrates between the 50 and 75 kDa bands as expected (lane 10). The three proteases can be seen migrating between the 25 and 37 kDa bands in the treatment lanes. Neither wild type hLYS nor the double mutant is degraded by the proteases, while BSA is fully degraded by all three. Similar results were obtained with both 5 µg/ml and 0.5 µg/ml protease treatments (data not shown), although BSA was only partially degraded by 0.5 µg/ml proteinase 3.

Mentions: In addition to thermal stability, resistance to human neutrophil proteases is an important parameter for pulmonary biotherapies, as neutrophil derived proteases can accumulate to micromolar concentrations in the infected and inflamed lung [25]. We therefore assessed proteolysis of the double mutant and wild type hLYS by human neutrophil elastase, cathepsin G, and proteinase 3. Our analysis shows that neither enzyme is susceptible to degradation by physiologically relevant concentrations of the three neutrophil proteases (Fig. 5). Combined with the thermal denaturation studies, these results indicate that the Arg101→Asp and Arg115→His mutations do not alter the double mutant's structural stability relative to wild type hLYS.


Crystal structure of a charge engineered human lysozyme having enhanced bactericidal activity.

Gill A, Scanlon TC, Osipovitch DC, Madden DR, Griswold KE - PLoS ONE (2011)

Proteolytic Susceptibility Analysis of Lysozymes.An SDS-PAGE gel was Coomassie stained for total protein. Lane 1 = Kaleidoscope prestained molecular weight standards. Lanes 2–5 = 900 ng of the engineered double mutant. Lane 2 is an untreated control, and lanes 3, 4, and 5 are 50 µg/ml treatments with human neutrophil elastase, cathepsin G, and proteinase 3, respectively. Lanes 6–9 are the wild type hLYS arrayed in a similar fashion. Lanes 10–13 are bovine serum albumin (BSA) arrayed in a similar fashion. The wild type hLYS and engineered double mutant migrate just below the 15 kDa band as expected (lanes 2–9). The BSA control migrates between the 50 and 75 kDa bands as expected (lane 10). The three proteases can be seen migrating between the 25 and 37 kDa bands in the treatment lanes. Neither wild type hLYS nor the double mutant is degraded by the proteases, while BSA is fully degraded by all three. Similar results were obtained with both 5 µg/ml and 0.5 µg/ml protease treatments (data not shown), although BSA was only partially degraded by 0.5 µg/ml proteinase 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049763&req=5

pone-0016788-g005: Proteolytic Susceptibility Analysis of Lysozymes.An SDS-PAGE gel was Coomassie stained for total protein. Lane 1 = Kaleidoscope prestained molecular weight standards. Lanes 2–5 = 900 ng of the engineered double mutant. Lane 2 is an untreated control, and lanes 3, 4, and 5 are 50 µg/ml treatments with human neutrophil elastase, cathepsin G, and proteinase 3, respectively. Lanes 6–9 are the wild type hLYS arrayed in a similar fashion. Lanes 10–13 are bovine serum albumin (BSA) arrayed in a similar fashion. The wild type hLYS and engineered double mutant migrate just below the 15 kDa band as expected (lanes 2–9). The BSA control migrates between the 50 and 75 kDa bands as expected (lane 10). The three proteases can be seen migrating between the 25 and 37 kDa bands in the treatment lanes. Neither wild type hLYS nor the double mutant is degraded by the proteases, while BSA is fully degraded by all three. Similar results were obtained with both 5 µg/ml and 0.5 µg/ml protease treatments (data not shown), although BSA was only partially degraded by 0.5 µg/ml proteinase 3.
Mentions: In addition to thermal stability, resistance to human neutrophil proteases is an important parameter for pulmonary biotherapies, as neutrophil derived proteases can accumulate to micromolar concentrations in the infected and inflamed lung [25]. We therefore assessed proteolysis of the double mutant and wild type hLYS by human neutrophil elastase, cathepsin G, and proteinase 3. Our analysis shows that neither enzyme is susceptible to degradation by physiologically relevant concentrations of the three neutrophil proteases (Fig. 5). Combined with the thermal denaturation studies, these results indicate that the Arg101→Asp and Arg115→His mutations do not alter the double mutant's structural stability relative to wild type hLYS.

Bottom Line: A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules.Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues.The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

ABSTRACT
Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

Show MeSH
Related in: MedlinePlus