Tandem repeats of Sushi3 peptide with enhanced LPS-binding and -neutralizing activities.
Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity. For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli. The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer). Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance. The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities. Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells. This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies.
Li
Changgui
C
Departments of Biological Sciences and Microbiology, 14 Science Drive 4, National University of Singapore, 117543 Singapore.
Ng
Miang Lon Patricia
ML
Zhu
Yong
Y
Ho
Bow
B
Ding
Jeak Ling
JL
D060829:Arthropod Proteins; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D019281:Dimerization; D004792:Enzyme Precursors; D006801:Humans; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D018805:Sepsis / Q000188:drug therapy; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D020080:Tandem Repeat Sequences
D060829:Arthropod Proteins; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C