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

Abstract

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 ␣-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 ␣-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. Apolipoprotein CII (apoCII)3 plays an important role in the metabolism of blood lipids as an activator of lipoprotein lipase (LPL) (1). Human apoCII is a 79-amino acid protein that is mainly expressed in liver and intestine (2). ApoCII appears in blood as a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins (3, 4). The mechanism for the activation has not been resolved in molecular detail, but the structures necessary for the activation reside in the C-terminal one-third of the apoCII sequence (5).Previous studies of the three-dimensional structure of apo-CII by NMR using full-length apoCII or an active fragment spanning residues 44 -79 in the presence of lipid mimetics like micelles of SDS or dodecylphosphatidylcholine have revealed that apoCII contains three ␣-helices spanning approximately residues 16 -38, 45-57, and 65-74 (6 -8). From earlier studies using synthetic peptide fragments of apoCII and secondary structure predictions it is known that the molecule is organized in two separate domains with different functions. The N-terminal part is involved in lipid binding by an amphipathic ␣-helix of the type found in all exchangeable apolipoproteins, whereas the C-terminal part (residues 56 -79) is responsible for activation of LPL (5, 9 -12). In several in vitro systems, the activation by the C-terminal fragments is comparable with the full-length apoCII, demon...

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces

“…In In addition, depending on their plasma concentrations, exchangeable apolipoproteins could absorb to the lipoprotein surface and increase surface pressure to such a degree that the lower-affi nity apolipoproteins are displaced ( 19,51 ). The two lipid-binding, class A, amphipathic ␣ -helices of apoC-II have similar hydrophobicity and length as those of apoC-I, but they differ in helical propensity and number of charged residues, such that apoC-I has a higher predicted lipoprotein affi nity than apoC-II ( 11,20 ). Notably, apoC-II is an activator of lipoprotein lipase and an important regulator of lipoprotein metabolism ( 11,20 ).…”

“…The two lipid-binding, class A, amphipathic ␣ -helices of apoC-II have similar hydrophobicity and length as those of apoC-I, but they differ in helical propensity and number of charged residues, such that apoC-I has a higher predicted lipoprotein affi nity than apoC-II ( 11,20 ). Notably, apoC-II is an activator of lipoprotein lipase and an important regulator of lipoprotein metabolism ( 11,20 ). We speculate that apoC-I could bind TAG-rich lipoproteins and increase local surface pressure above the retention pressure of apoC-II, thereby displacing apoC-II from the surface and inhibiting the catabolism of these lipoproteins by lipoprotein lipase.…”

Biochemistry and pathophysiology of intravascular and intracellular lipolysis

“…Without apo-CII, LPL is able to bind to lipoproteins, but catalytic activity is minimal (Olivecrona and Beisiegel 1997). Sequences within the C-terminal portion of apo-CII are essential for its interaction with LPL (Shen et al 2010). Sequences within the N-terminal portion of LPL have been implicated in apo-CII binding (Davis et al 1992), but C-terminal sequences also could play a role (Hill et al 1998).…”

A Pressure-dependent Model for the Regulation of Lipoprotein Lipase by Apolipoprotein C-II

“…Four residues (Tyr-63, Ile-66, Asp-69, and Gln-70) located on the same face of the C-terminal helix and exposed to solvent in NMR structures form a binding site for LPL (19,24,25). In vitro LPL activity assays showed that replacement of any of these four residues with alanine decreases the affinity of apoC-II for LPL and the catalytic activity of the LPL⅐apoC-II complex (24).…”