Heterologous expression of human uteroglobin/polychlorinated biphenyl-binding protein. Determination of ligand binding parameters and mechanism of phospholipase A2 inhibition in vitro.

High level expression of a human polychlorinated biphenyl-binding protein (hPCB-BP; also termed uteroglobin or CC10) was achieved in Escherichia coli. The recombinant protein (rhPCB-BP) constituted approximately 1% of total bacterial lysate proteins as judged from in vitro ligand binding assays using 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl. rhPCB-BP was purified to homogeneity in its native dimeric form. Saturation analysis experiments indicated a Kd of approximately 69 nM for the binding of 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl to rhPCB-BP. The average number of binding sites (Bmax) calculated from such experiments on purified rhPCB-BP was 49 nmol/mg of protein and is close to the theoretical value of 1 mol of ligand associating with 1 mol of dimeric protein. Purified rhPCB-BP was also found to cause a dose-dependent inhibition of the enzyme porcine pancreatic phospholipase A2 (PLA2) in vitro. Increasing the concentrations of calcium abolished the inhibition of PLA2 by rhPCB-BP, suggesting that the protein functions in vitro by sequestering Ca2+, an essential PLA2 cofactor. This notion was further supported by direct evidence that 45Ca2+ binds to rhPCB-BP. 1 mol of dimeric protein was also found to bind 2 mol of ruthenium red, an organic dye that detects Ca(2+)-binding proteins, with a Kd of 3 microM. This binding was inhibited by Ca2+, with an IC50 of 7 mM. Finally, it was demonstrated that the addition of a high affinity ligand for the protein had no effect on its ability to inhibit PLA2 under conditions of limiting concentrations of calcium, and the addition of Ca2+ did not affect the binding characteristics of the PCB ligand, suggesting that these two properties of the protein are independent. Our results strongly support the notion that ligand binding is a conserved feature of the homologous uteroglobin/PCB-BP/cc10 proteins in different species, whereas our results question the suggested role of these proteins as specific inhibitors of PLA2.