Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides

Three truncated fragments, harboring different sushi domains, namely, sushi 123, sushi1, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushil and 3 each has a high-affinity LPS binding site with K-d of 10(-9) to 10(-10) M. Positive cooperativity i n sushi123 resulted in a 1000-fold increase in K(d)2. The core LPS binding region of sushi and 3 reside in two 34-mer peptides, S1 and S3. A rigidly h eld disulfide-bonded structure is not essential but is important for LPS bi nding, as confirmed by a 100- to 10000-fold decrease in affinity. Both SI a nd S3 can inhibit LAL reaction and LPS-induced hTNF-alpha secretion with di fferent 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 Delta 1 an d S Delta 3 peptides exhibited increased LPS neutralization potential altho ugh its LPS binding affinities indicated only a 10-fold improvement. Hence, the structural difference of the four sushi peptides conferred different e fficiencies in LPS neutralization without altering their binding affinity f or LPS. Circular dichroism spectrometry revealed that the four peptides und erwent conformational change in the presence of lipid A, transitioning from a random coil to either an cu-helical or P-sheet structure. Two factors ar e critical for the sensitivity of Factor C to LPS: 1) the presence of multi ple binding sites for LPS on a single Factor C molecule; and 2) high positi ve cooperativity in LPS binding. The results showed that in the design of a n improved LPS binding and neutralizing peptide, charge balance of the pept ide is a critical parameter in addition to its structure.