Combined action of albumin and heparin regulates lipoprotein lipase oligomerization, stability, and ligand interactions.

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.

Calorimetric approach for comparison of Angiopoietin-like protein 4 with other pancreatic lipase inhibitors.

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.

An NMR and MD modeling insight into nucleation of 1,2-alkanediols: selective crystallization of lipase-catalytically resolved enantiomers from the reaction mixtures.

The work on developing a scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediols, complemented by crystallization of the pure enantiomers from the reaction mixtures, offered the possibility of a more detailed study of the aggregation of such diols. MD modeling, mass spectrometry, (1)H NMR, and DOSY studies provided a novel insight into the nucleation process. An efficient protocol for stereo- and chemoselective crystallization of (S)-1,2-dodecanediol and related compounds from the crude bioconversion mixtures was developed.

Synthesis and quantitative analysis of diastereomeric linked ester conjugates with remote stereocenters using high-field NMR and chiral HPLC.

A stereochemically safe high-yielding procedure for linking unprotected as well as protected hydroxycarboxylic acids to chiral secondary alcohols via glycolic acid linker is proposed. L-menthol has been linked with both enantiomers of mandelic, malic, and methoxyphenylacetic acid using bromo- or iodoacetyl group as a precursor of the glycolic acid linker. High-field nuclear magnetic resonance (NMR) and chiral high-performance liquid chromatography (HPLC) determination of high diastereomeric ratio (dr) (>99%) of the products bearing remote stereocenters was explored. Chiral HPLC allowed quantitation of the diastereomers up to dr 99.9/0.1. High-field NMR quantitation of the diastereomeric and parent alcoholic impurities in esters was demonstrated at the molar 0.3% and 0.03% levels, respectively. These analyses were done via comparison of integral intensities from major component (13)C satellites in (1)H or even in (13)C spectra to the (1)H or (13C signals of impurities. Despite lower sensitivity, the last option generally has much better selectivity. In this way the dynamic resolution is brought down by two orders.

Synthesis of deoxy sugar esters: a chemoenzymatic stereoselective approach affording deoxy sugar derivatives also in the form of aldehyde.

A chemoenzymatic synthesis of deoxy sugar esters is described. The synthesis is based on the O-alkylation of carboxylic acid with 2-bromo-5-acetoxypentanal. The method allows treatment of hydroxy carboxylic acids without protection of alcoholic hydroxyl groups. Several stereoisomeric deoxy sugar esters were resolved (up to ee or de > 98%) using a lipase-catalyzed acetylation of hemiacetals that in certain cases afforded deoxy sugar derivatives in the form of aldehydes. The stereochemistry of the reactions was determined by the NMR spectra of mandelic acid derivatives.