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 Tyr192 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 (approximately 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.
Ji
Y
Y
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois 61801, USA.
Jonas
A
A
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry*; D002918:Chymotrypsin / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D012996:Solutions; D014364:Tryptophan; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D004134:Dimyristoylphosphatidylcholine