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Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

Kiselar JG, Wang X, Dubyak GR, El Sanadi C, Ghosh SK, Lundberg K, Williams WM - PLoS ONE (2015)

Bottom Line: The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions.We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus

Comparison of deconvoluted tandom MS/MS spectra of untreated (a) vs modified (b) rhBD-2 peptide RYKQIGTCGLPGTK (23–36) after trypsin digestion of the rhBD-2 protein.The modified hBD-2 peptide was previously incubated in 100 μM MGO at 37°C for 72 h. The presence of the b1 ion with a +54 Da mass shift and unmodified doubly protonated y13 shows that modification of this peptide occurred at Arg23.
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pone.0130533.g003: Comparison of deconvoluted tandom MS/MS spectra of untreated (a) vs modified (b) rhBD-2 peptide RYKQIGTCGLPGTK (23–36) after trypsin digestion of the rhBD-2 protein.The modified hBD-2 peptide was previously incubated in 100 μM MGO at 37°C for 72 h. The presence of the b1 ion with a +54 Da mass shift and unmodified doubly protonated y13 shows that modification of this peptide occurred at Arg23.

Mentions: To determine the site of MGO modification, rhBD-2 was treated with dithiothreitol and then with iodoacetamide to reduce the three intramolecular disulfide bonds. Samples were then digested with trypsin and analyzed by LC-MS/MS as described in Methods. The ion signals at m/z 530.277 (2+), 467.556 (3+), 519.589 (3+), 526.951 (3+) and 566.294 (3+) that correspond to the unmodified peptides 1–10, 11–22, 11–23, 23–36 and 26–36, respectively, were observed by LC-MS analysis. The additional ion signals that correspond to a mass increase of +54 and +72 Da were detected only for peptides 11–23 and 23–36. Tandem MS analysis of these product ions revealed modification of the Arg23 residue by MGO (+72 Da) and by dehydrated MGO (+54 Da) adducts. Specifically, MS/MS analysis of triply protonated ion at m/z 544.955, corresponding to peptide 23–36 with a mass increase of +54 Da, produced a spectrum in which all the observed b-ions including a b1 ion were shifted by +54 Da (Fig 3b) relative to the corresponding control sample spectra of unmodified peptide 23–36 (Fig 3a). In contrast, all the observed y-ions, including doubly protonated y13 were unchanged. A similar fragmentation pattern was observed for triply protonated ion signal (at m/z 550.958) corresponding to peptide 23–36 with a mass shift of +72 Da. Furthermore, it was observed that all the b2-b7 and b10-ions were shifted by +72 Da (S3b Fig) while all the observed y-ions including doubly protonated y13 remained unmodified (S3a Fig). The MS/MS analysis of +54 and +72 Da adducts on peptide 11–23 of the rhBD2 protein showed a mass shift of +54 Da, corresponding to the modification of Arg22 (data not shown). An ion with a mass increase of +54 Da was the most prominent adduct observed for either the Arg22 or Arg23 peptide. Additionally, MGO adduction was observed on the N-terminus of the rhBD-2 protein in peptide 1–10. From the LC-MS/MS spectra of the doubly protonated ion (m/z 566.29), all observed b2-b4, b6 and b9 ions showed mass shifts of +72 Da, while observed y4 and y6-y9 ions remained unchanged. These observations indicate that modification of the peptide 1–10 by MGO (+72 Da) occurs at the N-terminal glycine (Gly1).


Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

Kiselar JG, Wang X, Dubyak GR, El Sanadi C, Ghosh SK, Lundberg K, Williams WM - PLoS ONE (2015)

Comparison of deconvoluted tandom MS/MS spectra of untreated (a) vs modified (b) rhBD-2 peptide RYKQIGTCGLPGTK (23–36) after trypsin digestion of the rhBD-2 protein.The modified hBD-2 peptide was previously incubated in 100 μM MGO at 37°C for 72 h. The presence of the b1 ion with a +54 Da mass shift and unmodified doubly protonated y13 shows that modification of this peptide occurred at Arg23.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526640&req=5

pone.0130533.g003: Comparison of deconvoluted tandom MS/MS spectra of untreated (a) vs modified (b) rhBD-2 peptide RYKQIGTCGLPGTK (23–36) after trypsin digestion of the rhBD-2 protein.The modified hBD-2 peptide was previously incubated in 100 μM MGO at 37°C for 72 h. The presence of the b1 ion with a +54 Da mass shift and unmodified doubly protonated y13 shows that modification of this peptide occurred at Arg23.
Mentions: To determine the site of MGO modification, rhBD-2 was treated with dithiothreitol and then with iodoacetamide to reduce the three intramolecular disulfide bonds. Samples were then digested with trypsin and analyzed by LC-MS/MS as described in Methods. The ion signals at m/z 530.277 (2+), 467.556 (3+), 519.589 (3+), 526.951 (3+) and 566.294 (3+) that correspond to the unmodified peptides 1–10, 11–22, 11–23, 23–36 and 26–36, respectively, were observed by LC-MS analysis. The additional ion signals that correspond to a mass increase of +54 and +72 Da were detected only for peptides 11–23 and 23–36. Tandem MS analysis of these product ions revealed modification of the Arg23 residue by MGO (+72 Da) and by dehydrated MGO (+54 Da) adducts. Specifically, MS/MS analysis of triply protonated ion at m/z 544.955, corresponding to peptide 23–36 with a mass increase of +54 Da, produced a spectrum in which all the observed b-ions including a b1 ion were shifted by +54 Da (Fig 3b) relative to the corresponding control sample spectra of unmodified peptide 23–36 (Fig 3a). In contrast, all the observed y-ions, including doubly protonated y13 were unchanged. A similar fragmentation pattern was observed for triply protonated ion signal (at m/z 550.958) corresponding to peptide 23–36 with a mass shift of +72 Da. Furthermore, it was observed that all the b2-b7 and b10-ions were shifted by +72 Da (S3b Fig) while all the observed y-ions including doubly protonated y13 remained unmodified (S3a Fig). The MS/MS analysis of +54 and +72 Da adducts on peptide 11–23 of the rhBD2 protein showed a mass shift of +54 Da, corresponding to the modification of Arg22 (data not shown). An ion with a mass increase of +54 Da was the most prominent adduct observed for either the Arg22 or Arg23 peptide. Additionally, MGO adduction was observed on the N-terminus of the rhBD-2 protein in peptide 1–10. From the LC-MS/MS spectra of the doubly protonated ion (m/z 566.29), all observed b2-b4, b6 and b9 ions showed mass shifts of +72 Da, while observed y4 and y6-y9 ions remained unchanged. These observations indicate that modification of the peptide 1–10 by MGO (+72 Da) occurs at the N-terminal glycine (Gly1).

Bottom Line: The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions.We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus