Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.     

Functional variants in the lipoprotein lipase gene and risk cardiovascular disease.

The current report is a quantitative review of the relationship between lipoprotein lipase gene variants and cardiovascular disease based on published population-based studies. Sixteen studies, representing 17,630 individuals, report allelic distribution for lipoprotein lipase gene variants among patients and control individuals. Patient outcomes included clinical cardiovascular disease events, documented coronary disease based on angiography, or intimal media thickening by B-mode ultrasonography. Mantel-Haenszel stratified analysis was used to compute a summary odds ratio and 95% confidence intervals for the association between rare allele in the lipoprotein lipase gene and disease status. Because of potential differing effects associated with different lipoprotein lipase variants, each lipoprotein lipase mutant allele was considered separately. The lipoprotein lipase D9N/-93G to T allele has a summary odds ratio of 2.03 (95% confidence interval 1.30-3.18), indicating a twofold increase in risk of coronary disease for carriers with this allelic variant. The summary odds ratio for the relationship of the rare lipoprotein lipase G188E variant with cardiovascular disease is 5.25 (95% confidence interval 1.54-24.29). The lipoprotein lipase N291S allele is associated with a marginal increase in cardiovascular disease (summary odds ratio 1.25, 95% confidence interval 0.99-1.60, P = 0.07). However, there is stronger evidence for a positive association in certain populations. The summary odds ratio for lipoprotein lipase S447X allele is 0.81 (95% confidence interval 0.65-1.0), which indicates a cardioprotective effect of this lipoprotein lipase gene variant. Thus, lipoprotein lipase gene variants are associated with differential susceptibility to cardiovascular disease.     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

Identification and characterization of the endothelial cell surface lipoprotein lipase receptor.

The hydrolysis of triglycerides in plasma lipoproteins is mediated by lipoprotein lipase (LPL) that is bound to vascular endothelial cells. The specific endothelial cell surface protein(s) with which LPL associates has not been characterized. To identify this LPL binding protein(s), radioiodinated cell surface proteins from cultured bovine aortic endothelial cells were chromatographed using bovine LPL-Sepharose. A single radioiodinated protein of apparent molecular mass 220 kDa was specifically retained by the gel and eluted with 0.4 M NaCl. A LPL-binding protein of similar size was obtained after metabolic labeling of the cellular proteoglycans with 35SO4, indicating that the 220-kDa protein is a proteoglycan. After heparitinase or nitrous acid treatments the molecular mass of the LPL-binding protein decreased to approximately 50 kDa, suggesting that it contains heparin sulfate chains. A 220-kDa protein from the basal cell surface was also identified using LPL-Sepharose chromatography. 125I-LPL was cross-linked to the endothelial cell surface using ethylene glycobis (succinimidylsuccinate). A single ligand-receptor complex, approximately 350 kDa, was obtained. Heparin and unlabeled LPL decreased the cross-linking of radioiodinated LPL to the cell surface receptor. To examine whether the receptor mediates the internalization of cross-linked 125I-LPL, cells containing 125I-LPL complexed to the surface were incubated at either 37 or at 4 degrees C. The amount of 125I-LPL internalized by the cells was 74% greater at 37 degrees C than at 4 degrees C. This suggested that LPL cross-linked to the receptor was internalized in a temperature-dependent manner. Thus, a 220-kDa heparan sulfate proteoglycan functions as an endothelial cell surface receptor for LPL.     

Determination of Borrelia surface lipoprotein anchor topology by surface proteolysis

We used a surface trypsinolysis assay to probe accessibility of the membrane-proximal N-terminal tether peptides of Borrelia surface lipoproteins OspA and Vsp1. Our findings with both wild-type and mutant proteins are only compatible with the anchoring of these surface lipoproteins in the outer leaflet of the outer spirochetal membrane.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

Lipoprotein lipase deficiency resulting from a nonsense mutation in exon 3 of the lipoprotein lipase gene.

The gene for erythroid 5-aminolevulinate synthase has been mapped to Xpter-Xq26 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to Xp21-Xq21, with the most likely location being on band Xp11.2. The mapping of the erythroid 5-amino-levulinate synthase gene to the X chromosome suggests that a defect in this gene may be the primary cause of X-linked sideroblastic anemia.     

Fate of lipoprotein lipase taken up by the rat liver. Evidence for a conformational change with loss of catalytic activity

When isolated rat livers were perfused with medium containing lipoprotein lipase, 40-60% was taken up during a single passage. This value was similar for lipoprotein lipase derived from culture medium of rat preadipocytes, and for lipoprotein lipase purified from bovine milk. It was also, similar, irrespective of the lipoprotein lipase concentration, at least up to 1 microgram/ml. Immediately following its uptake by the liver, a large fraction of the lipoprotein lipase could be released by heparin, but the magnitude of this fraction decreased with time. The enzyme lost its catalytic activity rather rapidly, but its degradation to acid-soluble products, or to larger fragments, was much slower. On heparin-agarose chromatography, the enzyme taken up by the liver eluted at a lower salt concentration than the original lipoprotein lipase preparation. This change in affinity for heparin suggests that the originally dimeric lipoprotein lipase had dissociated into monomers, in analogy to the findings in model experiments. It is suggested that the initial uptake of lipoprotein lipase occurs by binding to a polyanion at the liver cell surface. This is followed by endocytosis and dissociation of the enzyme from its heparan sulfate-like binding site. Acidification of the endosome may cause a conformational change in the lipase molecule with dissociation to inactive monomers, preceding ultimate proteolytic degradation.     

Hydrolysis of diacylglycerols by lipoprotein lipase.

Enantiomeric diacylglycerols were emulsified, mole for mole, with lyso(1-acyl) lecithin and were hydrolyzed with lipoprotein lipase in NH4Cl-beef serum albumin buffer at pH 8.6 after a brief incubation with delipidated rat serum. The enzyme was prepared from lyophilized and dialyzed bovine skim milk in a 4 percent solution. The course of hydrolysis for each set of enantiomers was determined by gas-liquid chromatography of the masses of the diacylglycerols remaining or monoacylglycerols released in the medium between 0 and 15 min. The majority of sets of sn-1,2- and 2,3-diacylglycerols, including an isotope-labeled true enantiomeric set which was assessed by mass spectrometry, demonstrated preference by the enzyme for lipolysis at position 1 but with less specificity than previously was shown in sn-triacylglycerol hydrolysis. The results preclude the possibility that the predominance of sn-2,3-diacylglycerol intermediates during triacylglycerol hydrolysis is due solely to a preferential breakdown of the 1,2-isomers and reinforce the conclusion that lipoprotein lipase is specific for position 1.     

The sequence of cDNA encoding lipoprotein lipase. A member of a lipase gene family

cDNA clones corresponding to the entire coding region of mature lipoprotein lipase were identified by antibody screening of a mouse macrophage library and sequenced. The predicted amino acid sequence indicates that the mature protein contains 447 amino acids with a molecular weight of 50,314. Comparison of the nucleotide and amino acid sequence with those of rat hepatic lipase and porcine pancreatic lipase reveals extensive homology among the enzymes, indicating that they are members of a gene family of lipases. Most striking is a conservation of five disulfide bridges in all three enzymes, strongly suggesting that the enzymes have similar overall folding patterns. Lipoprotein lipase is also shown to be extraordinarily conserved among mouse, human, and bovine species. The mRNA for lipoprotein lipase is abundant in heart and adipose tissue but is also present in a wide variety of other tissues. There are two major species of mRNA in mouse and human tissues examined, 3.6 and 3.4 kilobases (kb) in size. Rat tissues, on the other hand, contain only the 3.6-kb species while bovine tissues contain an additional 1.7-kb species.     

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.     

A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity.

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.     

Trp64----nonsense mutation in the lipoprotein lipase gene.

A lipoprotein lipase (LpL) gene defect has been identified, a G----A transition at nucleotide position 446 of exon 3, resulting in a premature termination codon (Trp----stop) at amino acid residue 64. This defect was identified in a Type I hyperlipoproteinemic subject with an amino acid residue 194 defect in the other allele. Plasma lipoprotein values as well as LpL mass and activity in postheparin plasma were determined in the subjects with the residue 64 defect and in other LpL-deficient heterozygotes. LpL mass levels in both the Type I and the other subject with a 64 LpL defect were markedly reduced. This may be explained by rapid degradation of LpL protein or decreased secretion from the 64 defective allele. Alternatively, the marked reduction or absence of mass associated with the 64 defect may be due to synthesis of a severely truncated protein which escapes immunologic detection.     

Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.     

Role of lipoprotein lipase in plasma triglyceride removal.

Intravenous injections of anti-lipoprotein lipase serunis quantitatively block the catabolism of very low density lipoprotein (VLDL) and portomicron triglyceride and specifically inhibit triglyceride transport into ovarian follicles. The immunological studies presented provide information on the site of action of lipoprotein lipase (LPL). In the anti-LPL serum-treated animals initial plasma triglyceride accumulation occurs at the time of antiserum injection. This instantaneous inhibition of triglyceride removal provides direct evidence that the functional LPL responsible for VLDL and portomicron triglyceride hydrolysis is located in sites within the plasma compartment readily accessible to immunoglobulins. In vitro immunological studies show that the adipose, heart, ovarian, and liver LPL share common immunological determinants. Biochemical studies on highly purified heart and adipose LPL suggest that these enzymes have identical protein moieties.     

Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing.

The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes. We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking. Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing. Larger insertions or deletions allowed the first step but progressively inhibited the second step. Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon.