rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21.
rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin. rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23. Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus. To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids. A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI). These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure). Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities. In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21. In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease. We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.
Horwitz
Arnold H
AH
XOMA (US) LLC, Berkeley, California 94710, USA.
Ammons
W Steve
WS
Bauer
Robert J
RJ
Dedrick
Russell
R
Nadell
Rossana
R
Williams
Robert E
RE
Liu
Pei-Syan
PS
D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D016470:Bacteremia; D001798:Blood Proteins / Q000378:metabolism* / Q000633:toxicity*; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine; D004195:Disease Models, Animal; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism* / Q000633:toxicity*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism / Q000633:toxicity; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D013211:Staphylococcus aureus / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C021623:bactericidal permeability increasing protein; D003545:Cysteine; D000409:Alanine