Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus
Human α-defensin 5 (HD5, HD5[subscript ox] to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial st...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society
2013
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| Online Access: | http://hdl.handle.net/1721.1/82575 https://orcid.org/0000-0002-6153-8803 |
| _version_ | 1826209825900462080 |
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| author | Wanniarachchi, Yoshitha A. Kaczmarek, Piotr Wan, Andrea Nolan, Elizabeth M. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Wanniarachchi, Yoshitha A. Kaczmarek, Piotr Wan, Andrea Nolan, Elizabeth M. |
| author_sort | Wanniarachchi, Yoshitha A. |
| collection | MIT |
| description | Human α-defensin 5 (HD5, HD5[subscript ox] to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys[superscript 3]-Cys[superscript 31], Cys[superscript 5]-Cys[superscript 20], Cys[superscript 10]-Cys[superscript 30]) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser[superscript hexa]], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S–S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5[subscript ox] against this Gram-positive bacterial strain. This observation supports the notion that the HD5[subscript ox] mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem.284, 29180−29192] and that the native disulfide array is a requirement for its activity against S. aureus. |
| first_indexed | 2024-09-23T14:31:42Z |
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| id | mit-1721.1/82575 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:31:42Z |
| publishDate | 2013 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/825752022-09-29T09:44:18Z Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus Wanniarachchi, Yoshitha A. Kaczmarek, Piotr Wan, Andrea Nolan, Elizabeth M. Massachusetts Institute of Technology. Department of Chemistry Wanniarachchi, Yoshitha A. Kaczmarek, Piotr Wan, Andrea Nolan, Elizabeth M. Human α-defensin 5 (HD5, HD5[subscript ox] to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys[superscript 3]-Cys[superscript 31], Cys[superscript 5]-Cys[superscript 20], Cys[superscript 10]-Cys[superscript 30]) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser[superscript hexa]], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S–S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5[subscript ox] against this Gram-positive bacterial strain. This observation supports the notion that the HD5[subscript ox] mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem.284, 29180−29192] and that the native disulfide array is a requirement for its activity against S. aureus. Massachusetts Institute of Technology. Biophysical Instrumentation Facility ((NSF- 0070319) Massachusetts Institute of Technology. Biophysical Instrumentation Facility (NIH GM68762) 2013-11-25T17:39:19Z 2013-11-25T17:39:19Z 2011-09 2011-08 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/82575 Wanniarachchi, Yoshitha A., Piotr Kaczmarek, Andrea Wan, and Elizabeth M. Nolan. “Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus.” Biochemistry 50, no. 37 (September 20, 2011): 8005-8017. https://orcid.org/0000-0002-6153-8803 en_US http://dx.doi.org/10.1021/bi201043j Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society PMC |
| spellingShingle | Wanniarachchi, Yoshitha A. Kaczmarek, Piotr Wan, Andrea Nolan, Elizabeth M. Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title | Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title_full | Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title_fullStr | Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title_full_unstemmed | Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title_short | Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus |
| title_sort | human defensin 5 disulfide array mutants disulfide bond deletion attenuates antibacterial activity against staphylococcus aureus |
| url | http://hdl.handle.net/1721.1/82575 https://orcid.org/0000-0002-6153-8803 |
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