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LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietin-like protein (ANGPTL)3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system.

Abstract GPIHBP1 is a membrane protein of endothelial cells that transports lipoprotein lipase (LPL), the key enzyme in plasma triglyceride metabolism, from the interstitial space to its site of action on the capillary lumen. An intrinsically disordered highly negatively charged N-terminal domain of GPIHBP1 contributes to the interaction with LPL. In this work, we investigated whether the plethora of heparin-binding proteins with positively charged regions found in human plasma affect this interaction. We also wanted to know whether the role of the N-terminal domain is purely non-specific and supportive for the interaction between LPL and full-length GPIHBP1, or whether it participates in the specific recognition mechanism. Using surface plasmon resonance, affinity chromatography, and FRET, we were unable to identify any plasma component, besides LPL, that bound the N-terminus with detectable affinity or affected its interaction with LPL. By examining different synthetic peptides, we show that the high affinity of the LPL/N-terminal domain interaction is ensured by at least ten negatively charged residues, among which at least six must sequentially arranged. We conclude that the association of LPL with the N-terminal domain of GPIHBP1 is highly specific and human plasma does not contain components that significantly affect this complex.

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.

Lipoprotein lipase (LPL), a crucial enzyme in the intravascular hydrolysis of triglyceride-rich lipoproteins, is a potential drug target for the treatment of hypertriglyceridemia. The activity and stability of LPL are influenced by a complex ligand network. Previous studies performed in dilute solutions suggest that LPL can appear in various oligomeric states. However, it was not known how the physiological environment, that is blood plasma, affects the action of LPL. In the current study, we demonstrate that albumin, the major protein component in blood plasma, has a significant impact on LPL stability, oligomerization, and ligand interactions. The effects induced by albumin could not solely be reproduced by the macromolecular crowding effect. Stabilization, isothermal titration calorimetry, and surface plasmon resonance studies revealed that albumin binds to LPL with affinity sufficient to form a complex in both the interstitial space and the capillaries. Negative stain transmission electron microscopy and raster image correlation spectroscopy showed that albumin, like heparin, induced reversible oligomerization of LPL. However, the albumin induced oligomers were structurally different from heparin-induced filament-like LPL oligomers. An intriguing observation was that no oligomers of either type were formed in the simultaneous presence of albumin and heparin. Our data also suggested that the oligomer formation protected LPL from the inactivation by its physiological regulator angiopoietin-like protein 4. The concentration of LPL and its environment could influence whether LPL follows irreversible inactivation and aggregation or reversible LPL oligomer formation, which might affect interactions with various ligands and drugs. In conclusion, the interplay between albumin and heparin could provide a mechanism for ensuring the dissociation of heparan sulfate-bound LPL oligomers into active LPL upon secretion into the interstitial space.