PROPERTIES OF AN N-TERMINAL PROTEOLYTIC FRAGMENT OF APOLIPOPROTEIN AI IN SOLUTION AND IN RECONSTITUTED HIGH-DENSITY-LIPOPROTEINS

Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein AI (apoAI), Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoAI and dipalmitoyl phosphatidylcholine or palmitoyloleoyl phosphatidylcholine by chymotrypsin, trypsin, elastase, and subtilisin generated a major fragment of 22 kDa. Under milder conditions proteolysis of lipid-free apoAI produced a fragment of similar size, The fragments shared the same N terminus as intact apoAI, and the chymotryptic fragment had a molecular weight of 22,384 as determined by electrospray ionization mass spectrometry, Thus the fragment consists of the N-terminal 192 amino acid residues of apoAI, and the region around Tyr(192) seems to be especially accessible to proteases. In aqueous solution the fragment, apoAI-(1-192), had an alpha-helix content similar to that of apoAI (similar to 52%) but existed only as monomers and dimers, ApoAI-(1-192) lysed dimyristoyl phosphatidylcholine liposomes slowly compared with apoAI but did form rHDL complexes with palmitoyloleoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine when prepared by the sodium cholate dialysis method, ApoAI-(1-192) rHDL exhibited sizes and size distributions distinct from apoAI: rHDL but displayed similar stability against denaturation, The isolated apoAI-(1-192) rHDLs retained a high ability to activate lecithin-cholesterol acyltransferase, comparable with the most effective apoAI rHDL, The results suggest that the C-terminal domain of apoAI is crucial for self-association and initial lipid binding but is not involved in specific lecithin-cholesterol acyltransferase activation.