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* Department of Biological Sciences,
Department of Microbiology, National University of Singapore, Singapore 117543
1Correspondence: Department of Biological Sciences, National University of Singapore, 10, Kent Ridge Crescent, Singapore 117543. E-mail: dbsdjl{at}nus.edu.sg
Three truncated fragments, harboring different sushi domains, namely,
sushi123, sushi1, and sushi3 domains, of Factor C were produced as
biologically active secreted recombinant proteins. Sushi1 and 3 each
has a high-affinity LPS binding site with Kd of
10-9 to 10-10 M. Positive cooperativity in
sushi123 resulted in a 1000-fold increase in
Kd2. The core LPS binding region of sushi1 and 3
reside in two 34-mer peptides, S1 and S3. A rigidly held
disulfide-bonded structure is not essential but is important for LPS
binding, as confirmed by a 100- to 10000-fold decrease in affinity.
Both S1 and S3 can inhibit LAL reaction and LPS-induced hTNF-
secretion with different potency. LAL assay revealed that at least two
molecules of S1 bind cooperatively to one LPS molecule, with Hill’s
coefficient of 2.42. The LPS binding by S3 is independent and
noncooperative. The modified S
1 and S3 peptides exhibited
increased LPS neutralization potential although its LPS binding
affinities indicated only a 10-fold improvement. Hence, the structural
difference of the four sushi peptides conferred different efficiencies
in LPS neutralization without altering their binding affinity for LPS.
Circular dichroism spectrometry revealed that the four peptides
underwent conformational change in the presence of lipid A,
transitioning from a random coil to either an -helical or ß-sheet
structure. Two factors are critical for the sensitivity of Factor C to
LPS: 1) the presence of multiple binding sites for LPS
on a single Factor C molecule; and 2) high positive
cooperativity in LPS binding. The results showed that in the design of
an improved LPS binding and neutralizing peptide, charge balance of the
peptide is a critical parameter in addition to its structure.—Tan,
N. S., Ng, M. L. P., Yau, Y. H., Chong, P. K. W., Ho, B., Ding, J. L. Definition of endotoxin binding
sites in horseshoe crab Factor C recombinant sushi proteins and
neutralization of endotoxin by sushi peptides.
Key Words: Limulus amebocyte lysate • lipid A • Drosophila S2 cells • LPS
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