Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23).
The 23-kDa recombinant amino-terminal bactericidal/permeability increasing protein fragment (rBPI23) has all of the antibacterial and antiendotoxin properties of the holoprotein. In the current studies, we have identified multiple active domains within rBPI23 with chemical and proteolytic cleavage fragments and with synthetic overlapping peptides. We also demonstrate a novel, high affinity heparin binding property for rBPI23, in addition to its established bactericidal and lipopolysaccharide binding properties. Cleavage fragments and synthetic, overlapping peptides of rBPI23 were analyzed for inhibition of the lipopolysaccharide-induced Limulus amebocyte lysate reaction, for bactericidal activity, and for heparin binding. Three separate, active domains were identified in amino acid regions 17-45, 65-99, and 142-169. A single synthetic peptide (85-99) was bactericidal. These results indicate that rBPI23 is comprised of three separate functional domains which contribute to the high affinity interaction of rBPI23 with Gram-negative bacteria. The individual activity of each domain and the cooperative interaction among domains provide the basis for developing rBPI23 analogues with increased biologic efficacy.
Little
R G
RG
Department of Immunology, XOMA Corporation, Berkeley, California 94710.
Kelner
D N
DN
Lim
E
E
Burke
D J
DJ
Conlon
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001665:Binding Sites; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D010450:Endopeptidases; D004926:Escherichia coli / Q000187:drug effects; D006493:Heparin / Q000737:chemistry; D006737:Horseshoe Crabs; D008033:Limulus Test; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C021623:bactericidal permeability increasing protein; D006493:Heparin; D010450:Endopeptidases; D003488:Cyanogen Bromide