rBPI(10—193) is secreted by CHO cells and retains the activity of rBPI21

Abstract

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(10193) 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.

Keywords

Human bactericidal/permeability-increasing protein recombinant fragments Gram-negative bacteria endotoxin BPI rBPI23 rBPI21 rBPI(10-193)

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