Chi-Sun Wang

The crystal structure of bovine bile salt activated lipase: insights into the bile salt activation mechanism.

BACKGROUNDnThe intestinally located pancreatic enzyme, bile salt activated lipase (BAL), possesses unique activities for digesting different kinds of lipids. It also differs from other lipases in a requirement of bile salts for activity. A structure-based explanation for these unique properties has not been reached so far due to the absence of a three-dimensional structure.nnnRESULTSnThe crystal structures of bovine BAL and its complex with taurocholate have been determined at 2.8 A resolution. The overall structure of BAL belongs to the alpha/beta hydrolase fold family. Two bile salt binding sites were found in each BAL molecule within the BAL-taurocholate complex structure. One of these sites is located close to a hairpin loop near the active site. Upon the binding of taurocholate, this loop becomes less mobile and assumes a different conformation. The other bile salt binding site is located remote from the active site. In both structures, BAL forms similar dimers with the active sites facing each other.nnnCONCLUSIONSnBile salts activate BAL by binding to a relatively short ten-residue loop near the active site, and stabilize the loop in an open conformation. Presumably, this conformational change leads to the formation of the substrate-binding site, as suggested from kinetic data. The BAL dimer observed in the crystal structure may also play a functional role under physiological conditions.

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: The identification of lipoprotein B as the main lipoprotein degradation product☆

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.

Inhibition of lipoprotein lipase by the receptor-binding domain of apolipoprotein E.

A synthetic peptide (residues 139–153) corresponding to the receptor‐binding domain of apolipoprotein E (ApoE) was tested for lipoprotein lipase (LPL) inhibitory properties. In systems using both natural and synthetic substrates, inhibition of LPL was observed. Using the synthetic substrate, 50% inhibition was observed at 50 μM while high concentrations completely inhibited LPL activity. These studies suggest an additional functional role for the receptor‐binding domain of ApoE — modulation of LPL activity.

Lipoprotein Particles in Hypertriglyceridemic States

The compositional and metabolic heterogeneity of operationally defined plasma lipoprotein classes (1-3) has necessitated the introduction of a classification system that utilizes apolipoproteins as specific markers for identifying and distinguishing discrete lipoprotein particles (1,4). In this system, lipoprotein particles are characterized and defined by their apolipoprotein composition (1,4). Studies on the quantification and distribution of apolipoproteins (4,5) have shown that apolipoprotein (Apo)B and ApoA (A-I 4- A-II) form two major groups of plasma lipoproteins. These two major lipoprotein groups may be separated (6) by immunoprecipitation or immunoaffinity chromatography of whole plasma (6). The use of these procedures results in the isolation of ApoA-containing lipoproteins free of ApoB. The fractionation of ApoA-containing lipoproteins into two major discrete lipoprotein particles LP-A-I and LP-A-I:A-II by immunoaffinity chromatography on an immunosorber with polyclonal antibodies to ApoA-II has already been described by Cheung and Albers (7). To identify discrete lipoprotein particles of the ApoB group of lipoproteins, we have developed a procedure based on sequential immunoprecipitation of ApoB-containing lipoproteins with polyclonal antisera to apolipoproteins B, E, C-III and, if necessary, C-II and C-I (6,8). The fractionation of very low density (VLDL, d < 1.006 g/ml) and two subtractions of low density (LDL.., d = 1.006-1.019 g/ml; LDL?, d = 1.019-1.063 g/ml lipoproteins from normolipidemic subjects by sequential immunoprecipitation showed that each of these density classes consists of a mixture of distinct lipoprotein particles including cholesterol ester-rich LP-B and triglyceride- rich LP-B:C-I:C-II:C-III:E (LP-B:C:E) and LP-B:C-I: C-II:C-III (LP-B:C) particles (8). The LP-B:C:E family of particles in some normolipidemic and hypercholesterolemic subjects also contained varying amounts of LP-B:E particles. In addition, small amounts of LP-B:C-I:E, LP-B:C-II, LP-C-III and LP-E particles were detected in some but not all-subjects or density classes. Each of the major ApoB-containing families of particles was shown to represent a polydisperse system of particles heterogeneous with respect to size, hydrated density, and lipid/protein ratio, but homogeneous with respect to the qualitative apolipoprotein composition.

Triglyceride-rich lipoprotein interactions with Lp(a).

We found a significantly reduced incidence of increased lipoprotein(a) (Lp(a)) levels in subjects with triglycerides (TG) greater than 150 mg/dl compared with those with TG levels lower than 150 mg/dl. This was the case in patients with angiographically documented coronary artery disease (CAD) and in subjects with no CAD. We explored the potential role of lipoprotein lipase (LPL) in mediating this relationship. Lp(a) and LDL2 exhibited a minimal effect on the rate constant for degradation of VLDL-TG by LPL (13% inhibition). Binding analyses indicated no differences between VLDL and LDL with respect to Lp(a) binding, and lipolysis only reduced binding by 30% at 75% degradation of VLDL-TG. Our study indicates that the inverse relationship between elevated plasma TG and Lp(a) levels is not caused by activation of LPL by Lp(a) either due to failure of Lp(a) to bind to VLDL or its lipolytic remnants. It is hypothesized that this relationship could stem from the enhanced clearance of TG-rich lipoproteins in individuals with higher levels of Lp(a) by receptor-mediated events.