Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-δ₁

It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃)-immobilized matrix was the δ₁ isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-δ₁ in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P₃ binding. The lysate was applied to Ins(1,4,5)P₃-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P₃ binding. The lysate was also purified to near homogeneity using a glutathione- Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-δ₁ consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P₃ binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-δ₁ which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P₃ binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-δ₁, which contains 6 basic amino acids, binds to an Ins(1,4,5)P₃-immobilized matrix. Moreover, Ins(1,4,5)P₃ binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-δ₁ is important for the binding of both Ins(1,4,5)P₃ and phosphatidylinositol 4,5-bisphosphate.