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Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4
Atsuo Maemoto1, Xiaoqing Qu, K Johan Rosengren
1Department of Pathology, College of Medicine, University of California, Irvine 92697-4800, USA.
The Journal of Biological Chemistry
|August 7, 2004
Summary
The disulfide bonds in mouse alpha-defensin Crp4 do not determine its antibacterial activity. Instead, these bonds protect Crp4 from degradation by matrix metalloproteinase-7 (MMP-7).
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Alpha-defensins are antimicrobial peptides characterized by a conserved tridisulfide array.
- The role of this invariant structural feature in alpha-defensin function remains incompletely understood.
- Mouse Paneth cell alpha-defensins, like cryptdin-4 (Crp4), require proteolytic activation by matrix metalloproteinase-7 (MMP-7).
Purpose of the Study:
- To investigate whether the tridisulfide array is essential for the bactericidal activity of mouse alpha-defensin Crp4.
- To determine the impact of disrupting the disulfide bonds on Crp4's susceptibility to MMP-7 degradation.
- To elucidate the functional significance of the Crp4 disulfide arrangement in the context of its activation and activity.
Main Methods:
- Site-directed mutagenesis was used to create Crp4 variants with altered disulfide bond connectivity.
- Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry confirmed the cysteine connectivities of the variants.
- In vitro bactericidal assays were performed to assess the antimicrobial activity of native and mutant Crp4.
- MMP-7 proteolysis assays were conducted to evaluate the susceptibility of native and mutant Crp4 and pro-Crp4 to degradation.
Main Results:
- Mutagenesis of disulfide bonds did not abolish Crp4's in vitro bactericidal activity; some variants showed enhanced activity.
- Native Crp4 and the defensin moiety of pro-Crp4 were resistant to MMP-7 degradation.
- All disulfide variants of Crp4 were extensively degraded by MMP-7.
- MMP-7 cleavage abolished the bactericidal activity of Crp4.
Conclusions:
- The tridisulfide array of mouse alpha-defensin Crp4 is not the primary determinant of its bactericidal activity.
- The disulfide arrangement is crucial for protecting Crp4 from proteolytic degradation by MMP-7.
- This protection is essential for maintaining Crp4's functional integrity and antimicrobial efficacy during its activation process.