BOTH N-TERMINAL 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 bacte ricidal/permeability-increasing protein (BPI), isolated after limited proteolysis, exhibits antibacterial and LPS-neutralizing activities eq ual 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 BP1-193 demonstrated the expected antibacte rial and LPS-binding activities, Deletion of the N-terminal 12 residue s did not diminish BPI function, However, further truncation either fr om the C-terminus to residue 169 (BPI1-169) or from the N-terminus (BP IDelta 15-56) yielded in vitro products with little or no LPS-binding activity and in vivo products that could not be recovered from the cul ture medium or cellular acid extracts, The possible role of cysteine-1 75 (the three cysteines in human BPI are at residues 132, 135, and 175 ) in BPI stability/function was examined by substitution of Cys(175) w ith serine, Recovery of C175S BPI from extracellular medium was reduce d 10-fold, and C175S BPI produced in vitro had little LPS-binding acti vity, Compared with wild-type holo-BPI and BPI1-193, BPI1-169, BPIDelt a 15-56, and C175S BPI showed increased susceptibility to cleavage by elastase in the region 1-193 (but not in the region 200-456), indicati ng conformational changes that may account for the loss of function, T hese 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 e ssential for structural stability and functional integrity.