Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.     

Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.     

A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma

Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant.     

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apo A-I and apo A-II inhibit hepatic triglyceride lipase from human postheparin plasma

Like α-amanitin, tRNA binds reversibly to yeast RNA polymerases A or B, in a 1:1 stoechiometry and inhibits enzyme B preferentially. Kinetic studies showed that the binding sites for the two inhibitors are completely independent. The dissociation constant of the enzyme-inhibitor complexes was determined. Only in the case of tRNA, it varied with the nature and concentration of the template. The stimulation factor P₃₇ does not interfere with the binding of both inhibitors.     

Endothelial lipase: a new member of the triglyceride lipase gene family

The triglyceride lipase gene family plays a central role in intestinal lipid absorption, energy homeostasis, lipoprotein metabolism, and atherosclerosis. A new member of this gene family, termed endothelial lipase, was recently reported. The presence of key functional motifs, the endothelial synthesis, the enzymatic profile, and the in-vivo metabolic effects of endothelial lipase suggest that, like other members of this gene family, endothelial lipase may play a role in energy delivery to tissues and in modulating lipoprotein metabolism, and could impact on atherogenesis.     

Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67,200 and 65,500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60,600 and 64,600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apolipoprotein C-II were 1.0 mM and 0.6 microM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum.     

Adipocyte triglyceride lipase expression in human obesity

We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.     

Effects of diet and age on lipoprotein lipase and hepatic triglyceride lipase activities in the rat

Plasma clearance of triglyceride-rich lipoproteins appears decreased in aged humans and rats and may be due to lowered activities of the lipases responsible for lipid degradation. This study was designed to examine differential effects of age and diet on lipoprotein lipase (LPL) activity of adipose and heart tissue and hepatic triglyceride lipase (HTGL) activity. LPL and HTGL activities were examined in 3- and 13-month-old Sprague-Dawley rats after they had consumed either a high-carbohydrate or a high-fat diet for 14 days. The data were analyzed for age and diet differences by two-way analysis of variance. Although animals in the two age groups consumed diets of equal caloric content, the older rats gained less weight. Rats on the high-carbohydrate diet consumed less calories and gained less weight than the fat fed rats in both age groups. Neither heart nor adipose tissue LPL activity differed when examined for age or diet. HTGL activity levels, while not affected by age, were higher in the carbohydrate fed rats (P = 0.014). Regardless of age group, fasting plasma cholesterol levels were significantly higher in the carbohydrate-fed rats than fat-fed rats (P = 0.002). Thus, the diet effect was much stronger than the age effect for HTGL and plasma cholesterol levels.     

Mechanism of inhibition of hepatic triglyceride lipase from human postheparin plasma by apolipoproteins A-I and A-II

The present data describe the mechanism of the inhibitory effects of human plasma apolipoproteins A-I and A-II on hydrolysis of triglyceride catalyzed by hepatic triglyceride lipase using a substrate of triolein particles stabilized with gum arabic in vitro. The experimental data could well be described by a model in which apolipoproteins bound to the surface of lipid substrate particles inhibited the enzyme reaction. The values of Km obtained were similar with or without inhibitors and the calculated saturation levels of apolipoprotein binding to the lipid were in good agreement with those obtained in independent binding experiments.     

Structure and polymorphic map of human lipoprotein lipase gene

Lipoprotein lipase (LPL) catalyzes the key step for the removal of triacylglycerol-rich lipoproteins from the circulation. In this paper, we report the cloning and structure of the normal human LPL gene, which was isolated in three overlapping lambda phage clones that span about 35 kilo bases (kb) of the genetic locus. The peptide coding region of the gene is approx. 23 kb in length and contains nine exons with intron sizes ranging from 0.7 to 8.7 kb. The entire 3' untranslated region is in the tenth exon. Specific sequences in this region support the hypothesis that two mRNA species found for human LPL are generated by differential utilization of polyadenylation signals. The first exon occurs in the 5' untranslated region and the region coding for the signal peptide. The second exon includes the protein domain coding for the N-linked glycosylation site that is required for the expression of enzyme activity. The fourth exon contains the region that was proposed as a lipid binding domain, the sixth for one putative heparin binding domain, and the eighth codes for a domain containing another N-linked glycosylation site. These results suggest that the unique structural and functional domains are confined to specific exons. The PvuII polymorphic site was located within the intron between exon 6 and 7 and the HindIII polymorphic site to the 3' flanking region. The location of these polymorphic sites suggests that the PvuII restriction fragment length polymorphism (RFLP) associated with lipase deficiency in a few Japanese kindred may be a linkage marker for a functional defect of LPL, while the HindIII RFLP associated with hypertriglyceridemia may be important for gene regulation of LPL.