Both N- and C-terminal regions of the bioactive N-terminal fragment of the neutrophil granule bactericidal/permeability-increasing protein are required for stability and function.

An N-terminal fragment (residues 1-199) of the 456-residue human bactericidal/permeability-increasing protein (BPI), isolated after limited proteolysis, exhibits antibacterial and LPS-neutralizing activities equal to or greater than those of holo-BPI. To assess minimal structural requirements for bioactivity, mutant species of BPI were expressed in vivo by transient transfection and in vitro by cellfree transcription/translation. BPI1-456 and BPI1-193 demonstrated the expected antibacterial and LPS-binding activities. Deletion of the N-terminal 12 residues did not diminish BPI function. However, further truncation either from the C-terminus to residue 169 (BPI1-169) or from the N-terminus (BPIdelta15-56) yielded in vitro products with little or no LPS-binding activity and in vivo products that could not be recovered from the culture medium or cellular acid extracts. The possible role of cysteine-175 (the three cysteines in human BPI are at residues 132, 135, and 175) in BPI stability/function was examined by substitution of Cys(175) with serine. Recovery of C175S BPI from extracellular medium was reduced 10-fold, and C175S BPI produced in vitro had little LPS-binding activity. Compared with wild-type holo-BPI and BPI1-193, BPI1-169, BPIdelta15-56, and C175S BPI showed increased susceptibility to cleavage by elastase in the region 1-193 (but not in the region 200-456), indicating conformational changes that may account for the loss of function. These findings suggest that the proteolytic N-terminal fragment of BPI corresponds closely to the minimum functional (antibacterial/anti-LPS) domain of BPI and that residues near both ends of this fragment are essential for structural stability and functional integrity.