Genetic variation and structure/function studies of human apolipoprotein A-IV in relation to atherosclerosis

Apolipoprotein A-IV (apoA-IV) is an anti-atherogenic lipoprotein, involved in the reverse cholesterol transport pathway and postprandial triglyceride clearance. The aim of this PhD was to characterise further the function of apoA-IV. Haplotype analysis of the APOA5-A4-C3 gene cluster revealed that the APOA4 T347S variant was associated with increased coronary heart disease (CHD) risk in the Northwick Park Heart II study [hazard ratio (HR) of 2.07 (95%CI 1.04 to 4.12)], independent of any effects on lipid levels. The S347 variant was also found to be associated with lower plasma apoA-IV levels (13.64+/-0.59 mg/dL) compared with carriers of the T347 allele (14.90+/-0.12 mg/dL, P=0.035) in the European Atherosclerosis Research Study I. Furthermore, amongst individuals with cardiovascluar disease (CVD) in the University College Diabetes and Cardiovascular Study, those homozygous for the S347 had significantly lower total anti-oxidant status compared to T347 carriers (31.2% 9.86 vs 42.5% 13.04, P=0.0024). Functional studies indicated that the pro-atherogenic role of apoA-IV-S347 could be due to its reduced antioxidant activity. Compared to wild type apoA-IV, apoA-IV-S347 decreased the time of 50% conjugated diene formation (T1/2 ) by 15% (P= 0.036) and apoA-IV-H360 increased Tm by 18% (P= 0.046). Potentially important residues of apoA-IV involved in LCAT activation were predicted by molecular modelling. Site directed mutagenesis was used to alter the polar face of helix 117-138 (R123Q, R134Q and N127R) imitating the three key arginine residues (R149, R153, R160) critical for apoA-I LCAT activation. On average the mutant apoA-IV proteins had reduced a-helicity compared to the wild type isoform in the lipid free state and limited differences in secondary structure once complexed with l-Palmitoyl-2-Oleoyl-sn-Glycero-3 Phosphocholine. These mutations did not alter the ability of apoA- IV to activate LCAT, indicating that the three critical arginine LCAT activation motif of apoA-I does not reside within helix 117-138 of apoA-IV