Inhibition of the phosphatidylinositol-specific phospholipase C from Bacillus cereus by a monoclonal antibody binding to a region with sequence similarity to eukaryotic phospholipases.
Bacterial phosphatidylinositol-specific phospholipases C (PI-PLC) display similar substrate specificity as their eukaryotic counterparts involved in signal transduction of insulin and Ca2(+)-mobilizing hormones, and are used in the study of the novel glycosylphosphatidylinositol-protein anchors (GPI-anchors). For the investigation of structure-function aspects of the PI-PLC secreted from Bacillus cereus cells, a panel of murine monoclonal antibodies was generated and shown to be specific for the PI-PLC polypeptide in enzyme-linked immunosorbent assays and Western blots. Two of the monoclonals inhibited reactions catalyzed by the bacterial enzyme in vitro: hydrolysis of phosphatidylinositol and the release of bovine erythrocyte acetylcholinesterase from its GPI-anchor. At saturating concentrations of inhibitory antibody only a few percent of the enzyme activity remained. The epitope recognized by one of the inhibitory antibodies, A72-24, was mapped by proteolytic digestion, protein sequencing, and Western blotting of the generated fragments. The data indicate that at least part of the epitope resides within an 8 kDa-stretch of the bacterial PI-PLC (Gln-45 - Lys-122). Essentially the same segment of the bacterial polypeptide has previously been shown to display limited amino acid sequence similarity with several eukaryotic PI-specific phospholipases C (Kuppe, A., Evans, L.M., McMillen, D.A. and Griffith, O.H. (1989) J. Bacteriol. 171, 6077-6083). The results reported here suggest that the conserved peptide of these enzymes may contain functionally important residues.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
A
A
Kuppe
K K
KK
Hedberg
J J
JJ
Volwerk
O H
OH
Griffith
D000110:Acetylcholinesterase / Q000097:blood; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001409:Bacillus cereus / Q000201:enzymology*; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase