Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.
Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.
Ridge
R J
RJ
Associates of Cape Cod, Inc., Falmouth, MA 02540, USA.
Paus
E J
EJ
Novitsky
T J
TJ
Ketchum
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006493:Heparin / Q000378:metabolism*; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D008070:Lipopolysaccharides; D010455:Peptides; C034705:antilipopolysaccharide factor (Limulus); D006493:Heparin