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-α) 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.
