Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis
Ji, Yong
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https://hdl.handle.net/2142/22853
Description
Title
Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis
Author(s)
Ji, Yong
Issue Date
1996
Doctoral Committee Chair(s)
Jonas, Ana
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Health Sciences, General
Language
eng
Abstract
Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I.
In aqueous solution the fragment, apoA-I(1-192), had a similar $\alpha$-helical content to apoA-I ($\sim$52%) but existed only as monomers and dimers. ApoA-I(1-192) lysed dimyristoyl PC (DMPC) liposomes slowly compared with apoA-I, but did form rHDL complexes with POPC or DPPC when prepared by the sodium cholate dialysis method. The isolated apoA-I(1-192) rHDLs activated lecithin cholesterol acyltransferase (LCAT), promoted cholesterol efflux from rat heptoma cells, and bound tightly to HeLa cells, indicating that the C-terminal 51 residues of apoA-I are crucial for self-association and initial lipid-binding but are not required for specific LCAT activation, cholesterol efflux or cell surface binding.
A highly efficient system was developed for overexpressing mature apoA-I and two C-terminal deletion mutants, apoA-I(1-164) and apoA-I(1-186), in E. coli. The recombinant apoA-I constituted more than 15% of the total cell protein. ApoA-I(1-164) was produced to a much lower level ($\sim$2-fold) than the other two proteins, suggesting that the C-terminal domain of apoA-I protects the protein from degradation. Essentially pure ($>$95%) apoA-I(1-164) and apoA-I(1-186) were obtained by reverse phase HPLC, giving a final yield for apoA-I(1-164) of $\sim$2 mg/liter culture.
ApoA-I(1-164) formed more dimers than monomers and had a lower $\alpha$-helical content than apoA-I(1-192); its Trp residues were more exposed to the aqueous environment. ApoA-I(1-164) exhibited decreased lipid-binding ability, indicating that the region between 165 and 192 is involved in lipid-protein interactions. Nonetheless, apoA-I(1-164) bound lipids cooperatively and formed POPC rHDL of distinct and defined sizes when prepared by the sodium cholate method.
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