An in vivo approach to the study of apolipoprotein A-I structure-function relationships.

Abstract

Apolipoprotein A-I (apoA-I) is a non-glycosylated 243 amino acid (aa) circulating plasma protein and the major constituent of high density lipoproteins (HDL). To study apoA-I in a physiologically relevant in vivo model, recombinant adenoviruses carrying cDNAs encoding for native human apoA-I (hapoA-I) and different apoA-I mutations were created. This is the first reported use of recombinant adenoviruses to study structure-function relationships of human apoA-I. In the first study, two functional domains within the central region of the protein (aa 100--186) were identified. The results establish that helix 6 (aa 144--165) is primarily responsible for activation of the enzyme lecithin:cholesterol acyltransferase (LCAT) while the more N-terminal half of the central domain, specifically the class Y helix 4 (aa 100--121), is important for lipid binding and/or stabilizing apoA-I on the surface of lipoproteins. A recombinant adenovirus was also produced encoding for a naturally occurring apoA-I point mutation with a Leu → Arg substitution at as 159. This apoA-I variant, known as apoA-I Finland (apoA-IFIN), causes an unexpected 80% reduction in HDL cholesterol (HDL-C) and apoA-I concentrations in heterozygous carriers. A combined in vitro, in vivo and ex vivo study demonstrated that this mutant has impaired activation of LCAT, is susceptible to proteolysis and has a decreased rate of secretion from primary hepatocytes. These dysfunctions appear to be responsible for the dominantly inherited hypoalphalipoproteinemia in heterozygous carriers of the apoA-I FIN mutation. Lastly, secretion of nascent HDL was monitored from primary hepatocytes. The recombinant adenovirus expressing hapoA-I was used to infect apoA-I deficient murine hepatocytes. In contrast to some previous studies, a significant pool of apoA-I was secreted with phospholipids (PL) and was heterogeneous in size. The role of this nascent HDL pool in determining plasma HDL-C concentrations is not known nor is the importance of other proteins in this process. The recent discovery of a major role for ATP binding cassette transporter A1 (ABCA1) in phospholipid (PL) and cholesterol efflux to apoA-I has led to the speculation that this protein may also shuttle lipids onto newly secreted apoA-I. The model described here will be useful to address this possibility.