Comparison of endotoxin antagonism of linear and cyclized peptides derived from limulus anti-lipopolysaccharide factor.
BACKGROUND
Limulus anti-lipopolysaccharide (LPS) factor (LALF) is a 102-amino acid LPS-binding protein from the horseshoe crab, Limulus polyphemus. The peptide includes the LPS-binding domain of holoLALF, yet it lacks the loop structure stabilized by disulfide or other covalent bonds that is a common motif in the LPS-binding regions of holo- LALF and several other LPS-binding proteins. Although it neutralizes LPS and is bactericidal against Pseudomonas aeruginosa, the LALF 28-54 portion of LALF is not protective in a murine model of intraperitoneal sepsis compared with holoLALF. We examined the effects of cyclizing this linear peptide to determine if this action would recapitulate the stable loop-type structure and enhance its LPS-neutralization and bactericidal activity in vitro.


METHODS
Cyclic LALF 28-54 was produced by oxidizing linear LALF 28-54. Each peptide, along with appropriate controls, was assayed for LPS neutralization using the chromogenic Limulus amebocyte lysate (LAL) assay and a bioassay to measure inhibition of LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha) by murine macrophages. Bactericidal activity against Pseudomonas aeruginosa was also assessed. Data were analyzed using a two-tailed Student t-test.


RESULTS
Polymyxin B and holoLALF exhibited potent endotoxin antagonism in both assays, as well as bactericidal activity. Concordant with prior studies, the linear form of LALF 28-54 exhibited either similar or a slightly lesser degree of activity in all assays. However, cyclization was associated with significantly diminished endotoxin neutralization in both assays (p < 0.05) and decreased bactericidal activity (p < 0.05) compared with linear LALF 28-54.


CONCLUSIONS
Whereas a synthetic linear peptide based on the endotoxin-binding region of holoLALF retained activity, cyclization was associated with a diminution in potency in vitro. We postulate that cyclization does not constrain the peptide in a manner that recreates the loop structure necessary for potent endotoxin antagonism.
Leslie
Daniel B
DB
Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Vietzen
Paul S
PS
Lazaron
Victor
V
Wasiluk
Karen R
KR
Dunn
David L
DL
D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D018929:Cell Culture Techniques; D003500:Cyclization; D004731:Endotoxins / Q000037:antagonists & inhibitors*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D007447:Invertebrate Hormones / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D011112:Polymyxin B / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000254:growth & development; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D004731:Endotoxins; D007447:Invertebrate Hormones; D014409:Tumor Necrosis Factor-alpha; C034705:antilipopolysaccharide factor (Limulus); D011112:Polymyxin B