Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr;22(3):218-29.
doi: 10.1177/1753425916631404. Epub 2016 Feb 15.

Carbamylated LL-37 as a Modulator of the Immune Response

Affiliations
Free PMC article

Carbamylated LL-37 as a Modulator of the Immune Response

Catalin Koro et al. Innate Immun. .
Free PMC article

Abstract

Carbamylation of lysine residues and protein N-termini is an ubiquitous, non-enzymatic post-translational modification. Carbamylation at sites of inflammation is due to cyanate formation during the neutrophil oxidative burst and may target lysine residues within the antimicrobial peptide LL-37. The bactericidal and immunomodulatory properties of LL-37 depend on its secondary structure and cationic nature, which are conferred by arginine and lysine residues. Therefore, carbamylation may affect the biological functions of LL-37. The present study examined the kinetics and pattern of LL-37 carbamylation to investigate how this modification affects the bactericidal, cytotoxic and immunomodulatory function of the peptide. The results indicated that LL-37 undergoes rapid modification in the presence of physiological concentrations of cyanate, yielding a spectrum of diverse carbamylated peptides. Mass spectrometry analyses revealed that theN-terminal amino group of Leu-1 was highly reactive and was modified almost instantly by cyanate to generate the predominant form of the modified peptide, named LL-37(C1) This was followed by the sequential carbamylation of Lys-8, Lys-12, and Lys-15 to yield LL-37(C8), and Lys-15 to yield LL-37(C12,15) Carbamylation had profound and diverse effects on the structure and biological properties of LL-37. In some cases, anti-inflammatory LL-37 was rapidly converted to pro-inflammatory LL-37.

Keywords: Carbamylation; LL-37; immunomodulation.

Figures

Figure 1
Figure 1. The quantity of carbamylated amino acid residues increases with elevated KCNO concentration
LL-37 was incubated for 3 hours with increasing amounts of KCNO. A) 0 mM KCNO, B) 10 mM KCNO, C) 50 mM KCNO. The samples were analysed by liquid chromatography (LC)-MS/MS and the m/z values corresponding to LL37 with 0 to 7 carbamylated amino acid residues were extracted (colour code according to legend). The KCNO mediated carbamylation results in a heterogeneous population of carbamylated LL-37 as seen by the appearance of multiple peaks. D) The N-terminal leucine (L1) and lysine at position 8 on LL-37 are highly accessible to carbamylation. LL-37 were incubated with 10 mM or 50mM KCNO for 1 hour at 37°C. The position of modification was determined using NanoESI-MS/MS after V8 digestion. Spectral count were calculated by taking the sum of the number of spectra matching the identified peptides.
Figure 2
Figure 2. CD spectra of native and carbamylated LL-37
The peptide concentration were 10 μM. 50% TFE buffer contained 50% TFE diluted in 10mM Sodium Phosphate buffer. The plasma buffer contained 113 mM NaCl, 24 mM NaHCO3, 0.6 mM MgCl2, 1.3 mM CaCl2, 3.9mM KCl. (A) Spectra are the mean of two (50% TFE buffer) or three (plasma buffer) independent experiments. (B) Difference of predicted α-helical content between the peptide analogues in 50% TFE buffer at 190–260 nm. (C) Difference of predicted α-helical content between the peptide analogues in Plasma buffer at 190–260 nm. (B–C) Data are expressed by the mean ± SD. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test. p<0.05, **p<0.01; ***p<0.001. TFE, trifluorethanol.*
Figure 3
Figure 3. Carbamylation abrogate the antimicrobial activity of LL-37
(A–C) The number of colony forming units formed after incubation with (A) B. subtilis, (B) E. coli and (C) S. aureus. All bacterial species were incubated with carbamylated or native LL-37. Bacterial growth in the presence of native LL-37 was set at 100%. Data are expressed as the mean ± SD. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons post-test. *p<0.05, **p<0.01; ***p<0.001; whereas #p <0.05; ##p< 0.01; ###p<0.001 as compared to (A–C) no peptide or(D–E) LPS.
Figure 4
Figure 4. Carbamylation does not impair LPS binding capacity of the LL-37
Human monocyte derived macrophages were incubated with 100 ng/ml LPS in the absence or presence of variously modified peptides. The levels of (A) TNF-α and (B) IL-6 in the supernatants were then measured and LL-37+LPS was set to 100%. Data are expressed as the mean ±SD. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons post-test. *p<0.05, **p<0.01; ***p<0.001; whereas #p <0.05; ##p< 0.01; ###p<0.001 as compared to LPS.
Figure 5
Figure 5. Carbamylation of LL-37 reduce the binding capacity to apolipoprotein A1
Surface plasmon resonance characteristics for the interaction between ApoA1 and carbamylated and native LL-37 at a peptide concentration of 1μM. RU, resonance units
Figure 6
Figure 6. Carbamylation decrease chemotactic potential of LL-37 peptide
Analysis of chemotaxis using 20 μM peptide concentration. Neutrophils from 4 healthy volunteers were treated with either native or modified peptide. Extracted values for each individual’s speed, velocity, chemotactic index and resultant vector length were analysed for statistical difference. The midline of each box represents median. The statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons post-test: *p < 0.05; **p < 0.01; ***p < 0.001; whereas #p < 0.05; ##p < 0.01; ###p < 0.001 as compared to native peptide.
Figure 7
Figure 7. Carbamylation significantly affect the hemolytic capacity of LL-37
Erythrocytes were incubated for 2 hours with LL-37 analogues at a peptide concentration ranging from 1–20 μM. The hemolytic activity of the peptides was evaluated by recording the release of hemoglobin at 405 nm from human erythrocytes upon incubation. Data represent 4 individual experiments and are expressed as means ±SD. The statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparisons post-test: *p < 0.05; **p < 0.01; ***p < 0.001 when compared with LL-37 at a peptide concentration of 2 μM.

Similar articles

See all similar articles

Cited by 5 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback