Human granulocytes express a 55-kDa lipopolysaccharide-binding protein on the cell surface that is identical to the bactericidal/permeability-increasing protein.

Abstract

Several LPS-binding proteins have been identified on the surface of human granulocytes (polymorphonuclear leukocyte (PMN)). We describe a plasma-membrane associated ca. 55-kDa LPS-binding protein of human PMN that is indistinguishable from the bactericidal/permeability-increasing protein (BPI). To detect LPS-binding proteins on the cell surface, PMN were biotinylated before detergent solubilization and incubation with LPS-coated beads. Several biotinylated proteins bound to LPS-coated beads but not to uncoated beads and were characterized after elution with detergent by SDS-PAGE and western blotting using streptavidin-horseradish peroxidase. The spectrum of biotinylated proteins binding to and eluting from LPS-coated beads increased as the number of beads incubated with PMN lysate increased. However, at all concentrations of beads a 55-kDa protein was a dominant component of the eluate. Binding of the 55-kDa protein to LPS-coated beads was inhibited by lipid A, and both homologous and heterologous LPS, but not by peptidoglycan. Similar amounts of biotinylated 55-kDa LPS-binding protein were detected on PMN from patients with paroxysmal nocturnal hemoglobinuria who lacked membrane bound CD14, a known ca. 55-kDa plasma membrane-associated LPS-binding protein, indicating that the recovered biotinylated protein is not CD14. Several pieces of evidence, however, do indicate that the 55-kDa surface protein is BPI: 1) flow cytometry of PMN after labeling with rabbit anti-BPI serum and FITC-labeled goat anti-rabbit IgG revealed immunoreactive surface molecules on resting PMN and, in increased amounts, on PMN stimulated with FMLP or TNF; 2) This antiserum specifically and quantitatively inhibited binding of the biotinylated 55-kDa species to LPS-coated beads; 3) both BPI and the 55-kDa protein migrated as a doublet during SDS-PAGE and were both converted to single migrated species after N-glycosidase F treatment; 4) chemical cleavage of the biotinylated protein and native BPI with N-chlorosuccinimide yielded the same fragments. Thus, we have positively identified BPI as a LPS-binding protein on the surface of PMN. The role of this potent antibacterial, endotoxin neutralizing protein on the surface of PMN remains to be established.