Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.</p

Site-directed Mutagenesis of Apolipoprotein CII to Probe the Role of Its Secondary Structure for Activation of Lipoprotein Lipase*

Apolipoprotein CII (apoCII) is a necessary activator for lipoprotein lipase (LPL). We had identified four residues (Tyr-63, Ile-66, Asp-69, and Gln-70), presumably contained in an α-helix, as a potential binding site for LPL. We have now used structure prediction, mutagenesis, and functional assays to explore the functional role of the secondary structure in this part of apoCII. First, mutants were generated by replacements with proline residues to disturb the helical structure. Activation by mutant G65P was reduced by 30%, whereas mutant S54P retained activation ability. Mutants V71P and L72P should be located outside the LPL-binding site, but V71P was totally inactive, whereas activation by L72P was reduced by 65%. Insertion of alanine after Tyr-63, changing the position of the putative LPL-binding site in relation to the hydrophobic face of the α-helix, also severely impeded the activation ability, and a double mutant (Y63A/I66A) was completely inactive. Next, to investigate the importance of conserved hydrophobic residues in the C-terminal end of apoCII, Phe-67, Val-71, Leu-72, and Leu-75 were exchanged for polar residues. Only F67S showed dramatic loss of function. Finally, fragment 39–62, previously claimed to activate LPL, was found to be completely inactive. Our data support the view that the helical structure close to the C-terminal end of apoCII is important for activation of LPL, probably by placing residues 63, 66, 69, and 70 in an optimal steric position. The structural requirements for the hydrophobic face on the back side of this helix and further out toward the C terminus were less stringent