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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

Effect of MGO adduction to rhBD-2 on chemoattraction for CEM cells.Inset, untreated rhBD-2 shows a concentration-dependent optimum in chemoattraction. Fluorescence units are proportional to migrated cell number due to labeling of migrating cell DNA by the CyQuant probe. Lower graph, chemoattractive function is reduced by incubating rhBD-2 with 100 uM MGO for 72 h at 37ଌ. SDF-1 is positive control for chemotaxis of CEM cells. Data is presented as the Mean ± S.E.M. for N1 = 3, N2 = 3 experiments * *: p = 0.05).
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pone.0130533.g005: Effect of MGO adduction to rhBD-2 on chemoattraction for CEM cells.Inset, untreated rhBD-2 shows a concentration-dependent optimum in chemoattraction. Fluorescence units are proportional to migrated cell number due to labeling of migrating cell DNA by the CyQuant probe. Lower graph, chemoattractive function is reduced by incubating rhBD-2 with 100 uM MGO for 72 h at 37ଌ. SDF-1 is positive control for chemotaxis of CEM cells. Data is presented as the Mean ± S.E.M. for N1 = 3, N2 = 3 experiments * *: p = 0.05).

Mentions: Because the hBD-2 peptide exhibits chemoattractive properties for CD4+ memory T cells [35, 36], we tested the ability of MGO-induced adducts of the rhBD-2 peptide to alter chemotactic function using the T-lymphoblastoid CEM-SS cell line. Both SDF-1 (positive control) and untreated (unadducted) rhBD-2 exhibited strong chemoattraction for our CEM test cells, with a direct correlation between rhBD-2 concentration and level of chemoattraction (Fig 5, inset). MGO-induced adduction of rhBD-2 resulted in a loss of chemotactic function that was observed beginning at rhBD-2 concentrations of 20 μg/ml and reaching statistical significance at 40 μg/ml (Fig 5). These results demonstrate that adduction of specific residues contributes to reduced chemotactic function by rhBD-2.


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)

Effect of MGO adduction to rhBD-2 on chemoattraction for CEM cells.Inset, untreated rhBD-2 shows a concentration-dependent optimum in chemoattraction. Fluorescence units are proportional to migrated cell number due to labeling of migrating cell DNA by the CyQuant probe. Lower graph, chemoattractive function is reduced by incubating rhBD-2 with 100 uM MGO for 72 h at 37ଌ. SDF-1 is positive control for chemotaxis of CEM cells. Data is presented as the Mean ± S.E.M. for N1 = 3, N2 = 3 experiments * *: p = 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526640&req=5

pone.0130533.g005: Effect of MGO adduction to rhBD-2 on chemoattraction for CEM cells.Inset, untreated rhBD-2 shows a concentration-dependent optimum in chemoattraction. Fluorescence units are proportional to migrated cell number due to labeling of migrating cell DNA by the CyQuant probe. Lower graph, chemoattractive function is reduced by incubating rhBD-2 with 100 uM MGO for 72 h at 37ଌ. SDF-1 is positive control for chemotaxis of CEM cells. Data is presented as the Mean ± S.E.M. for N1 = 3, N2 = 3 experiments * *: p = 0.05).
Mentions: Because the hBD-2 peptide exhibits chemoattractive properties for CD4+ memory T cells [35, 36], we tested the ability of MGO-induced adducts of the rhBD-2 peptide to alter chemotactic function using the T-lymphoblastoid CEM-SS cell line. Both SDF-1 (positive control) and untreated (unadducted) rhBD-2 exhibited strong chemoattraction for our CEM test cells, with a direct correlation between rhBD-2 concentration and level of chemoattraction (Fig 5, inset). MGO-induced adduction of rhBD-2 resulted in a loss of chemotactic function that was observed beginning at rhBD-2 concentrations of 20 μg/ml and reaching statistical significance at 40 μg/ml (Fig 5). These results demonstrate that adduction of specific residues contributes to reduced chemotactic function by rhBD-2.

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