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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

MALDI-TOF MS spectra obtained from native (untreated) rhBD-2 peptide (A) vs MGO-treated peptide (B). Native peptide exhibits singly and doubly protonated molecular ionic species at m/z 4326.6 and 2164.5, respectively. Exposure of rhBD-2 (20 ng/μl) to 100 μM MGO for 72 h resulted in additional peaks for both the singly and doubly protonated ionic species. Singly protonated ions (shaded area) show m/z increases of + 54 Da (m/z 4381.7), +72 Da (m/z 4399.7), and +126 Da (m/z 4453.7), suggesting adduction of dehydrated, intact, and dehydrated + intact MGO molecular species, respectively.
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pone.0130533.g001: MALDI-TOF MS spectra obtained from native (untreated) rhBD-2 peptide (A) vs MGO-treated peptide (B). Native peptide exhibits singly and doubly protonated molecular ionic species at m/z 4326.6 and 2164.5, respectively. Exposure of rhBD-2 (20 ng/μl) to 100 μM MGO for 72 h resulted in additional peaks for both the singly and doubly protonated ionic species. Singly protonated ions (shaded area) show m/z increases of + 54 Da (m/z 4381.7), +72 Da (m/z 4399.7), and +126 Da (m/z 4453.7), suggesting adduction of dehydrated, intact, and dehydrated + intact MGO molecular species, respectively.

Mentions: We used MALDI-TOF MS to determine if incubation of rhBD-2 with MGO or GO could induce mass changes in the peptide reflective of MGO- or GO-derived adducts. Untreated rhBD-2 incubated at 37°C in phosphate-buffered saline (PBS) for 72 h exhibited peaks at m/z of 4327.6 and 2164.5 (Fig 1A). These peaks corresponded to singly (1+) and doubly (2+) protonated rhBD-2 ionic species. Incubation of rhBD-2 in 100 μM MGO for 72 h, however, resulted in the appearance of additional peaks that corresponded in mass to molecular species of MGO (Fig 1B). These mass changes were equivalent to the mass of rhBD-2 protein adducted by intact MGO (m/z 4399.6), a dehydrated species (m/z 4381.7), or by a combined intact + dehydrated molecular species (m/z 4453.9) of MGO (Fig 1B, shaded area). Incubations less than 72 h and ranging from 2 h to 48 h also resulted in the appearance of additional peaks with increasing intensities at m/z of 4399.5, 4435.7 and 4453.6 (Fig 2a–2c). These masses corresponded to increases of +72 (intact), +108 (2 dehydrated), and +126 (1 dehydrated + 1 intact) representing MGO-derived adduction to rhBD-2 peptide. We observed a change in both peak profile and intensity when the molar concentration of MGO was reduced from 100 μM to 10 μM with incubation times of 24, 48 and 72 h (Fig 2d–2f). At 2 h incubation times, peaks corresponding to MGO-derived adducts were indistinguishable from background (data not shown). GO was far less reactive with the rhBD-2 peptide than MGO (S2 Fig). Nevertheless, mass increases of +40 (m/z 4367.7) and + 58 (m/z 4385.5), corresponding to dehydrated and intact GO-derived adducts were detected after incubation of the peptide to 100 μM GO for 48 and 72 h.


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)

MALDI-TOF MS spectra obtained from native (untreated) rhBD-2 peptide (A) vs MGO-treated peptide (B). Native peptide exhibits singly and doubly protonated molecular ionic species at m/z 4326.6 and 2164.5, respectively. Exposure of rhBD-2 (20 ng/μl) to 100 μM MGO for 72 h resulted in additional peaks for both the singly and doubly protonated ionic species. Singly protonated ions (shaded area) show m/z increases of + 54 Da (m/z 4381.7), +72 Da (m/z 4399.7), and +126 Da (m/z 4453.7), suggesting adduction of dehydrated, intact, and dehydrated + intact MGO molecular species, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526640&req=5

pone.0130533.g001: MALDI-TOF MS spectra obtained from native (untreated) rhBD-2 peptide (A) vs MGO-treated peptide (B). Native peptide exhibits singly and doubly protonated molecular ionic species at m/z 4326.6 and 2164.5, respectively. Exposure of rhBD-2 (20 ng/μl) to 100 μM MGO for 72 h resulted in additional peaks for both the singly and doubly protonated ionic species. Singly protonated ions (shaded area) show m/z increases of + 54 Da (m/z 4381.7), +72 Da (m/z 4399.7), and +126 Da (m/z 4453.7), suggesting adduction of dehydrated, intact, and dehydrated + intact MGO molecular species, respectively.
Mentions: We used MALDI-TOF MS to determine if incubation of rhBD-2 with MGO or GO could induce mass changes in the peptide reflective of MGO- or GO-derived adducts. Untreated rhBD-2 incubated at 37°C in phosphate-buffered saline (PBS) for 72 h exhibited peaks at m/z of 4327.6 and 2164.5 (Fig 1A). These peaks corresponded to singly (1+) and doubly (2+) protonated rhBD-2 ionic species. Incubation of rhBD-2 in 100 μM MGO for 72 h, however, resulted in the appearance of additional peaks that corresponded in mass to molecular species of MGO (Fig 1B). These mass changes were equivalent to the mass of rhBD-2 protein adducted by intact MGO (m/z 4399.6), a dehydrated species (m/z 4381.7), or by a combined intact + dehydrated molecular species (m/z 4453.9) of MGO (Fig 1B, shaded area). Incubations less than 72 h and ranging from 2 h to 48 h also resulted in the appearance of additional peaks with increasing intensities at m/z of 4399.5, 4435.7 and 4453.6 (Fig 2a–2c). These masses corresponded to increases of +72 (intact), +108 (2 dehydrated), and +126 (1 dehydrated + 1 intact) representing MGO-derived adduction to rhBD-2 peptide. We observed a change in both peak profile and intensity when the molar concentration of MGO was reduced from 100 μM to 10 μM with incubation times of 24, 48 and 72 h (Fig 2d–2f). At 2 h incubation times, peaks corresponding to MGO-derived adducts were indistinguishable from background (data not shown). GO was far less reactive with the rhBD-2 peptide than MGO (S2 Fig). Nevertheless, mass increases of +40 (m/z 4367.7) and + 58 (m/z 4385.5), corresponding to dehydrated and intact GO-derived adducts were detected after incubation of the peptide to 100 μM GO for 48 and 72 h.

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