BINDING OF PHOSPHOLIPID TRANSFER PROTEIN (PLTP) TO APOLIPOPROTEINS A-I AND A-II - LOCATION OF A PLTP BINDING DOMAIN IN THE AMINO-TERMINAL REGION OF APOA-I

The interaction of plasma phospholipid transfer protein (PLTP) with HD L has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-me diated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule . To study the PLTP/apolipoprotein The interaction we used a solid pha se ligand binding assay, the ELISA technique, and apoA-I and apoA-II a ffinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a conce ntration-dependent manner, and the binding could be displaced by prein cubating the PLTP sample with purified apolipoproteins. To determine w hich portion of apoA-I is recognized by PLTP, we coated ELISA plates w ith either recombinant full-length apoA-I or three shortened apoA-I fo rms sequentially truncated from the C-terminus. To characterize the PL TP binding ability of the C-terminal region of apoA-I, we used both C- terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. T o further confirm the identity of the binding region, we probed the in teraction with a polyclonal and several monoclonal anti-apoA-I antibod ies. The antibodies that inhibited the interaction between PLTP and ap oA-I were directed towards apoA-I epitopes localized between amino aci ds 27-141. The polyclonal antibody R33, and the monoclonal antibody A- I-1 (epitope between amino acids 27-48) were most effective and reduce d PLTP binding by 70%. These results show that PLTP binds to both apoA -I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.