Limits...
Structure-Dependent Immune Modulatory Activity of Protegrin-1 Analogs.

Zughaier SM, Svoboda P, Pohl J - Antibiotics (Basel) (2014)

Bottom Line: Protegrins are porcine antimicrobial peptides (AMPs) that belong to the cathelicidin family of host defense peptides.Protegrin-1 (PG-1), the most investigated member of the protegrin family, is an arginine-rich peptide consisting of 18 amino acid residues, its main chain adopting a β-hairpin structure that is linked by two disulfide bridges.We report on the immune modulatory activity of PG-1 and its analogs in neutralizing bacterial endotoxin and capsular polysaccharides, consequently inhibiting inflammatory mediators' release from macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA ; Laboratories of Microbial Pathogenesis, Atlanta Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA.

ABSTRACT

Protegrins are porcine antimicrobial peptides (AMPs) that belong to the cathelicidin family of host defense peptides. Protegrin-1 (PG-1), the most investigated member of the protegrin family, is an arginine-rich peptide consisting of 18 amino acid residues, its main chain adopting a β-hairpin structure that is linked by two disulfide bridges. We report on the immune modulatory activity of PG-1 and its analogs in neutralizing bacterial endotoxin and capsular polysaccharides, consequently inhibiting inflammatory mediators' release from macrophages. We demonstrate that the β-hairpin structure motif stabilized with at least one disulfide bridge is a prerequisite for the immune modulatory activity of this type of AMP.

No MeSH data available.


PG-1 analogs neutralized meningococcal capsular polysaccharide (CPS) polymers activity and inhibited IL-1β release. CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS. IL-1β was released from human macrophage-like THP-1 cells induced overnight with meningococcal CPS polymers (25 μg/mL) pre-incubated with or without 2 μg/mL of PG-1 or its derivatives for 30 min at 37 °C. IL-1β release was measured by ELISA. Error bars represent ±SD from the mean of duplicate measurements. This experiment is representative of two independent experiments. Methylated: N-methylated tyrosine or phenylalanine derivatives; S–S: disulfide bridges.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4472440&req=5

Figure 5: PG-1 analogs neutralized meningococcal capsular polysaccharide (CPS) polymers activity and inhibited IL-1β release. CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS. IL-1β was released from human macrophage-like THP-1 cells induced overnight with meningococcal CPS polymers (25 μg/mL) pre-incubated with or without 2 μg/mL of PG-1 or its derivatives for 30 min at 37 °C. IL-1β release was measured by ELISA. Error bars represent ±SD from the mean of duplicate measurements. This experiment is representative of two independent experiments. Methylated: N-methylated tyrosine or phenylalanine derivatives; S–S: disulfide bridges.

Mentions: Specifically, we investigated the immune modulatory response towards completely or partially linearized analogs, testing the importance of disulfide bridges of PG-1 (Compound 1). We also tested the importance of intra-/inter-chain hydrogen bonding potential, as H-bonding is presumed to be a major driving force behind oligomerization of PG-1 at bacterial membranes [63]. The analogs used in this work are categorized into four groups based on their common structural characteristics (Figure 1 and Table 1). Group I derivatives 1–4 include the native PG-1 (1) and all maintain disulfide bridge connectivity. As expected, the D-amino acid analog 2 has very similar activity to the parent peptide 1 and effectively neutralized meningococcal LOS and CPS bioactivity, leading to inhibition of TNFα (Figures 2 and 3), IL-1β (Figures 4 and 5), and nitric oxide (Figures 6 and 7) release even when used at low dose of 2 μg/mL [52]. Peptide 3 contains a dodecanoyl (C12) moiety at the N-terminus of 1, making it more hydrophobic. Compared to PG-1, acylation did not improve immune modulatory activity against meningococcal LOS (Figures 2 and 4) and was even less effective against neutralizing CPS polymers (Figures 3 and 5). This observation is contrary to LL-37, for which Nα-terminus acylation markedly enhanced its immune modulatory activity against LOS and CPS polymers [57]. Furthermore, acylation of the cathepsin G (CG) peptide, which has an α-helical structure, led to enhancement of its activity [64,65]. The length of fatty acyl chain matters and C12 chain length were found to be optimal for enhancing CG peptide activity when compared to shorter or longer chain lengths ranging from C4–C18, as previously shown [66]. Therefore, acylation of peptides with a β-hairpin structure like PG-1 may potentially interfere with peptide oligomerization, which is a very refined process prerequisite to its activity [32]. Analog 4 contains in place of its six arginine residues homoarginine residues, thus making it more apolar as compared to the parent peptide 1 while retaining the positively-charged guanidinium groups. Side-chain guanidinium groups of Arg residues were shown to be critical for PG-1 interaction with bacterial surface phosphate moieties [67,68]. It can be seen that the replacement of arginines with bulkier homoarginines resulted in immune modulatory derivative 4, the most potent that we have tested so far against meningococcal LOS and CPS (Figures 2, 3, 6, and 7). We note that the number of arginines was found to be critical for protegrin antimicrobial activity [69]. Tang et al. showed that reducing the number of arginines dramatically reduced antibacterial activity due to a reduction in membrane insertion and the inability of arginine to electrostatically bind to the phosphate groups on lipid A [55,69,70]. The presence of phosphate groups is critical for interaction with cationic peptides, and elimination of these negative charges by phosphoethanolamine or carbohydrate residues replacement confers resistance to AMPs like polymyxin B and LL-37 [71]. PG-1 is also shown to bind more efficiently to LPS from Pseudomonas aeruginosa as compared to LPS from Burkholderia cepacia [72]. The reduced binding due to substitution of phosphate head groups with 4-aminoarabinose in B. cepacia lipid A is thought to be the major determinant of resistance [72].


Structure-Dependent Immune Modulatory Activity of Protegrin-1 Analogs.

Zughaier SM, Svoboda P, Pohl J - Antibiotics (Basel) (2014)

PG-1 analogs neutralized meningococcal capsular polysaccharide (CPS) polymers activity and inhibited IL-1β release. CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS. IL-1β was released from human macrophage-like THP-1 cells induced overnight with meningococcal CPS polymers (25 μg/mL) pre-incubated with or without 2 μg/mL of PG-1 or its derivatives for 30 min at 37 °C. IL-1β release was measured by ELISA. Error bars represent ±SD from the mean of duplicate measurements. This experiment is representative of two independent experiments. Methylated: N-methylated tyrosine or phenylalanine derivatives; S–S: disulfide bridges.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472440&req=5

Figure 5: PG-1 analogs neutralized meningococcal capsular polysaccharide (CPS) polymers activity and inhibited IL-1β release. CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS. IL-1β was released from human macrophage-like THP-1 cells induced overnight with meningococcal CPS polymers (25 μg/mL) pre-incubated with or without 2 μg/mL of PG-1 or its derivatives for 30 min at 37 °C. IL-1β release was measured by ELISA. Error bars represent ±SD from the mean of duplicate measurements. This experiment is representative of two independent experiments. Methylated: N-methylated tyrosine or phenylalanine derivatives; S–S: disulfide bridges.
Mentions: Specifically, we investigated the immune modulatory response towards completely or partially linearized analogs, testing the importance of disulfide bridges of PG-1 (Compound 1). We also tested the importance of intra-/inter-chain hydrogen bonding potential, as H-bonding is presumed to be a major driving force behind oligomerization of PG-1 at bacterial membranes [63]. The analogs used in this work are categorized into four groups based on their common structural characteristics (Figure 1 and Table 1). Group I derivatives 1–4 include the native PG-1 (1) and all maintain disulfide bridge connectivity. As expected, the D-amino acid analog 2 has very similar activity to the parent peptide 1 and effectively neutralized meningococcal LOS and CPS bioactivity, leading to inhibition of TNFα (Figures 2 and 3), IL-1β (Figures 4 and 5), and nitric oxide (Figures 6 and 7) release even when used at low dose of 2 μg/mL [52]. Peptide 3 contains a dodecanoyl (C12) moiety at the N-terminus of 1, making it more hydrophobic. Compared to PG-1, acylation did not improve immune modulatory activity against meningococcal LOS (Figures 2 and 4) and was even less effective against neutralizing CPS polymers (Figures 3 and 5). This observation is contrary to LL-37, for which Nα-terminus acylation markedly enhanced its immune modulatory activity against LOS and CPS polymers [57]. Furthermore, acylation of the cathepsin G (CG) peptide, which has an α-helical structure, led to enhancement of its activity [64,65]. The length of fatty acyl chain matters and C12 chain length were found to be optimal for enhancing CG peptide activity when compared to shorter or longer chain lengths ranging from C4–C18, as previously shown [66]. Therefore, acylation of peptides with a β-hairpin structure like PG-1 may potentially interfere with peptide oligomerization, which is a very refined process prerequisite to its activity [32]. Analog 4 contains in place of its six arginine residues homoarginine residues, thus making it more apolar as compared to the parent peptide 1 while retaining the positively-charged guanidinium groups. Side-chain guanidinium groups of Arg residues were shown to be critical for PG-1 interaction with bacterial surface phosphate moieties [67,68]. It can be seen that the replacement of arginines with bulkier homoarginines resulted in immune modulatory derivative 4, the most potent that we have tested so far against meningococcal LOS and CPS (Figures 2, 3, 6, and 7). We note that the number of arginines was found to be critical for protegrin antimicrobial activity [69]. Tang et al. showed that reducing the number of arginines dramatically reduced antibacterial activity due to a reduction in membrane insertion and the inability of arginine to electrostatically bind to the phosphate groups on lipid A [55,69,70]. The presence of phosphate groups is critical for interaction with cationic peptides, and elimination of these negative charges by phosphoethanolamine or carbohydrate residues replacement confers resistance to AMPs like polymyxin B and LL-37 [71]. PG-1 is also shown to bind more efficiently to LPS from Pseudomonas aeruginosa as compared to LPS from Burkholderia cepacia [72]. The reduced binding due to substitution of phosphate head groups with 4-aminoarabinose in B. cepacia lipid A is thought to be the major determinant of resistance [72].

Bottom Line: Protegrins are porcine antimicrobial peptides (AMPs) that belong to the cathelicidin family of host defense peptides.Protegrin-1 (PG-1), the most investigated member of the protegrin family, is an arginine-rich peptide consisting of 18 amino acid residues, its main chain adopting a β-hairpin structure that is linked by two disulfide bridges.We report on the immune modulatory activity of PG-1 and its analogs in neutralizing bacterial endotoxin and capsular polysaccharides, consequently inhibiting inflammatory mediators' release from macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA ; Laboratories of Microbial Pathogenesis, Atlanta Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA.

ABSTRACT

Protegrins are porcine antimicrobial peptides (AMPs) that belong to the cathelicidin family of host defense peptides. Protegrin-1 (PG-1), the most investigated member of the protegrin family, is an arginine-rich peptide consisting of 18 amino acid residues, its main chain adopting a β-hairpin structure that is linked by two disulfide bridges. We report on the immune modulatory activity of PG-1 and its analogs in neutralizing bacterial endotoxin and capsular polysaccharides, consequently inhibiting inflammatory mediators' release from macrophages. We demonstrate that the β-hairpin structure motif stabilized with at least one disulfide bridge is a prerequisite for the immune modulatory activity of this type of AMP.

No MeSH data available.