title: Conversion of pig pancreas phospholipase A2 by protein engineering into enzyme active against Escherichia coli treated with the bactericidal/permeability-increasing protein.
abstract: Phospholipases A2 (PLA-2) are conserved enzymes that can vary widely in their activity toward certain biological targets. Activity of PLA-2 toward Escherichia coli treated with the bactericidal/permeability-increasing protein (BPI) of granulocytes has been detected only in "Group II" PLA-2 (lacking Cys11-Cys77) and correlates with overall basicity and the presence of a cluster of basic amino acids within a variable surface region near the NH2 terminus (including residues 6, 7, 10, 11, and 15). We now show that of five pancreatic PLA-2 ("Group I" enzymes) tested from different species of mammals, the human enzyme that is most basic both globally (pI 8.7) and locally (Arg-6, Lys-7, and Lys-10) is active toward BPI-treated E. coli (approximately 1-2% activity of the most active Group II PLA-2) whereas the other four PLA-2 are essentially inactive (less than 0.1%). The cDNA of the pig pancreatic PLA-2 (pI 6.4; Arg-6, Ser-7, Lys-10) has been modified by site-specific mutagenesis and the wild-type and mutant PLA-2 have been expressed in and purified from either E. coli or Saccharomyces cerevisiae to determine more precisely the structural determinants of PLA-2 activity toward BPI-treated E. coli. The single substitution of lysine (or arginine) for Ser-7 transformed the pig pancreatic PLA-2 into an active enzyme toward BPI-treated E. coli possessing 25-50% the activity of the human PLA-2. Additional modifications to increase global basicity (increase in net charge up to +4) caused a further (up to 2-fold) increase in activity. All mutant PLA-2 still containing Ser-7 possessed little or no activity toward BPI-treated E. coli. Changes in activity toward BPI-treated E. coli were accompanied by parallel changes in enzyme binding to this target. In contrast, substitution of lysine (or arginine) for Ser-7 caused little or no alteration of enzyme activity toward either autoclaved E. coli or egg yolk lipoproteins indicating no major effects on the catalytic properties of the PLA-2. This study demonstrates directly the role of NH2-terminal basic residues in the action of PLA-2 on BPI-treated E. coli and suggests that these properties mainly facilitate PLA-2 binding to this biological target.
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York 10016.
authors.forename: J
authors.initials: J
authors.lastname: Weiss
authors.forename: G
authors.initials: G
authors.lastname: Wright
authors.forename: A C
authors.initials: AC
authors.lastname: Bekkers
authors.forename: C J
authors.initials: CJ
authors.lastname: van den Bergh
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D008565:Membrane Proteins; C021623:bactericidal permeability increasing protein; D010741:Phospholipases A; D054467:Phospholipases A2