An analysis of mutations underlying lipoprotein lipase (LPL) deficiency in France

Monday 10 October 1994: Poster Abstracts Lipases

12581Binding to heparan sulfate is an obligatory initial step in lipoprotein Iipase (LPL) catabolii by HepG2 and other cell cultures $&y&E, Olivecrona T, Bengtsson-Olivecrona G, Vlodavsky I, Levkovitz H, Avner R, Eisenberg S, Inst. of Lipid and Atheroscle-

67

same experimental conditions and in the studied range of concentrations, repurified cholesterol had no effect. These results indicating a marked effect of oxysterols on LPL activity warrant further study.

rosis Res., The Chaim Sheba Med. Center, Tel Hashomer, Israel 52621

Apo E inhibition of LPL-mediated lipolysis of triglyceride-rich emulsions resembling cbvlomicrons wPCN, Van Berkel TJC, Div. of Biopha-kceuttcs, Leiden-

The object was to determine the nature of LPL interactions with liver parenchyma cells as compared to other cell cultures. The binding, cell association and degradation of ‘251-labeled bovine milk LPL was examined in HepG2 cells, normal and LDL receptor-negative human fibrohlasts and CHO cells. The binding, cell association and degradation of ‘251-labeled LPL by the different cells showed similar values irrespective of source and origin. HepG2 cells were characterized by a high capacity to bind and degrade LPL and an extremely high sensitivity to heparin; the binding and degradation were inhibited by treatment of the cells with Na chlorate and heparinase (but not chondroitinase). To further clarify the role of heparan sulfate in LPL-cell interactions we compared the metabolism of LPL in wild-type and mutant, heparan sulfate-deficient, CHO cells. The latter showed low capacity to bind and degrade LPL, about 10% that of the wild type cells. In another set of experiments we asked whether LPL interaction with HepG2 cells is affected by triglyceride-rich lipoproteins. Unlabeled LPL dramatically enhanced the metabolism of radioiodinated VLDL, but unlabeled VLDL had no effect on radioiodinated LPL metabolism in these cells. These results indicate that (a) the high capacity of the liver to metabolize LPL is dependent on initial binding of the enzyme to heparan sulfate present on the surface of parenchyma liver cells and (b) LPL interaction with hepatocytes is independent of the metabolism of triglyceride-rich lipoproteins.

Amsterdam Center for Drug Res., Sylvius Lab., PO Box 9503, 2300 RA Leiden, The Netherlands

Oxysterols decrease in vitro lipoprotein lipaseinduced lipolysis of triglyceride-rich substrates Blache D, Polette A, INSERM, Lab. de Biochimie des Lipoprote-

12591

ines, Univ. de Bourgogne, Fact&e’ de Medecine, d’Arc, F-21033 Dijon, France

12601

Apo E is an important determinant for the liver uptake of TG-rich emulsions by the remnant receptor. The aim of this study was to assess an additional role of apo E as modulator of the metabolism of TG-rich lipoproteins. Glycerol tri[3H]oleate-laheled, 82-nm TG-rich emulsions (1 .O mg/ml TG) were incubated with 4.0 &nl LPL in 60 mg/rnl free fatty acid-free BSA, 5% (v/v) heat-inactivated rat serum and 0.1 M Tris-HCI (pH 8.5). Rapid hydrolysis of triglycerides occurred, as determined by photon correlation spectroscopy and 13H]oleate extraction. Addition of apo E caused a marked concentration-dependent non-competitive inhibition of [3H]oleate release (9.5% residual LPL activity at 6Opglml apo E). Apo A-I, isolated from human HDL, did not affect the lipolysis rate at similar concentrations as apo E. The inhibitory effect of apo E was only slightly affected by modification of its lysine residues by reductive methylation. In contrast, selective modification of a&tine residues by 1,2-cyclohexadione in borate buffer completely cancelled the inhibitory effect of apo E. Lactoferrin, which contains an arginine cluster similar to that of apo E and therefore effectively blocks the liver uptake of chylomicrons and beta-VLDL, did not inhibit LPL. We conclude that apo E can effectively inhibit the LPL mediated hydrolysis of emulsion triglycerides. Since these emulsions mimic chylomicrons, we suspect that in addition to its role in receptor-recognition, apo E could also modulate the LPLmediated hydrolysis of triglycerides.

7, Bd Jeanne

A number of studies have shown that cholesterol oxidation products such as oxysterols found in LDL modified by metals or vascular cells are readily taken up by macrophages to form foam cells within the vascular wall. Oxysterols can be found in foodstuffs and be absorbed and transported in plasma lipoproteins. Oxyster01s have been predominantly identified in plasma in the LDL and fractions. We describe here the effect of several oxysterols on lipolysis mediated in vitro by lipoprotein lipase (LPL). Two substrates have been used: oxysterols incorporated into an artificial substrate made of triglyceride-rich liposomes, and human VLDL enriched in vitro with oxysterols. The oxysterols were individually incorporated into phospholipid vesicles with sterols: phospholipid molar ratios ranging from 0.01 to 0.3. Oxysterols were also incorporated into human VLDL by in vitro incubation and analyzed by capillary gas-liquid chromatography. The oxysterol enrichment was less than 10% of the endogenous cholesterol. Bovine milk LPL was used and assayed in the presence of apolipoprotein C-II as activator for the artificial substrate. For incubations with the triolein-rich liposomes, the release of radiolabeled oleate by LPL was measured using the silicon dioxide technique. For incubations in the presence of the oxysterolenriched VLDL, the release of fatty acids was assayed with a commercial kit (Wako). Oxysterols elicited a marked decrease of the lipolysis of both substrates. The reduction of lipoprotein lipase activity by oxysterols was concentration-dependent. Oxysterols oxidized in the ring or the side chain were both able to inhibit LPL-induced lipolysis, 25-hydroxycholesterol being the most potent. Under the

An analysis of mutations underlying lipoprotein lipase (LPL) deficiency in France w*, Gag& E, De Gennes JL*, Ma Y, Forsythe I, Liu MS, Zhang H, Dairou F, Lagarde JP*, Benlian P*, Hayden MR,

12611

*Service d ‘endocrinologie-mCtabolisme, Hopital de la Pitie, 83 bd de l’hopital, 75013 Paris, France; Univ. of British Columbia, Vancouver. Canada

Complete LPL deficiency associated with chylomicronemia is an uncommon autosomal recessive disorder caused by many different LPL gene mutations in patients of different origins. Our aim was to establish the molecular basis for LPL deficiency in French subjects. Twelve unrelated French probands with familial chylomicronemia were studied. LPL deficiency was confirmed by measurements of enzyme mass and activity in post-heparin plasma. We screened for the presence of mutations on genomic DNA by Southern blotting, PCR, enzymatic restriction, SSCP and AS0 hybridization. Final identification of mutations was obtained by genomic sequencing. Thirteen different mutations accounted for 19/24 alleles. Among the missense mutations four were new (Asp156 --f His, Gly188 -+ Arg, Ser259 + Arg, Let1286 --t Pro). Interestingly, the Asp156 + His and the Gly188 --f Arg represented mutations affecting different nucleotides of a codon with previously described substitutions. The Gly188 + Glu, Arg243 + Cys, Asp250 + Asn mutations were previously reported. All were located in exons 5 and 6, and were associated with a detectable but an inactive protein in plasma, as assessed by in vitro sitedirected mutagenesis. A 1 bp insertion in exon 2 and two 2 bp

Atherosclerosis X, Montreal, October 1994

Monday 10 October 1994: Poster Abstracts Lipases

68

deletions in exon 3 represented novel frameshift mutations. We have also found a 4 bp deletion in exon 4, a partial duplication of exon 6 and a deletion of exon 9 as previously reported. All these gene rearrangements were expressed as null alleles. Our data provide further evidence that molecular defects of the LPL gene are heterogeneous and that mutations resulting in a catalytically defective protein are clustered in exons 5 and 6 of the LPL gene. cDNA cloning of lysosomal acid lipase (LAL) and its mutation in rats with inherited LAL deficiency mH, Matsubara S, Yoshidome H, Yoshida H, Kuriyama M, Osame M, 3rd Dept. of hat. Med., Kagoshima Univ. Sch. of

12621

Med., 8-35-l

Sakuragaoka. Kagoshima 890, Japan

Lysosomal acid lipase (LAL) is a key enzyme in lipid metabolism and atherosclerosis. We previously reported a rat model of Wolman disease (rWD), a congenital defect of LAL (J Lipid Res 1990; 31: 1605). In this study, we cloned rat LAL (rLAL) cDNA and investigated abnormal LAL gene expression in rWD. The rLAL was cloned from a cDNA library of a normal rat using human LAL cDNA as a probe. The hybridized products were subcloned into Ml3 vector and sequenced. We also analyzed a cDNA library from the rWD liver using the rLAL cDNA. The rLAL cDNA consisted of 3150 bp nucleotides including an 1194 bp open reading frame and 3 poly A signals at the 3’end. The deduced amino acid sequence, having a 397-mer sequence, showed 79.9% homology with human LAL and had two identical functional domains at the same sites as human LAL. Northern blot analysis revealed that the rLAL mRNA in normal rat was 3.2 kb in length, while the rLAL mRNA in rWD was 1.4 kb, with a 1.7 kb deletion. The rWD LAL cDNA had the same sequence as the rLAL cDNA from the 5’ untranslated region to nt 1101, a 60 bp replacement from nt 1102 to nt 1061 with pfiy A signal, and a 1.7 kb deletion, indicating the substitution of Ile to Asn, and 368Pro to stop codon, and 29 amino acids missing. Genomic Southern blot analysis disclosed a large deletion at the 3’end. These results suggest that the C-terminus of rLAL may be essential for the enzyme activity. Production and characterization of a specific monoclonal antibody against lipoprotein lipase aF, Murthy MRV, Marcotte B, LRvesque G, Deshaies Y, Lupien PJ, Julien P, QuJbec Lipid Res. Centre, Luval Univ. Med.

12631

Centre, Ste-Foy, QC, Canada GI V 4G2

A fragment of human lipoprotein lipase (LPL) cDNA (405 bp, 5’ terminal end) was cloned in an expression vector (PET-3c) and was used to transform DE3-BL2N bacteria. The fusion peptide of -17 kDa was injected into mice and the spleen cells were fused with mouse myeloma SP2 cells. The hybridoma extracts were tested against semi-purified bovine milk LPL and also against rat, mouse and human plasma LPL, by Western blot analysis. The LPL-specific monoclonal antibody (MAb) thus selected was purified by affinity chromatography and labeled with peroxidase. Using this MAb in a sandwich enzyme-linked immunoflow assay (ELIFA), it was possible to quantitate LPL mass in both monomeric and dimeric forms in tissue extracts. The LPL immunoreactive mass of heart and adipose tissue extracts paralleled LPL activities in these tissues. The MAb did not inhibit the LPL enzymatic activity which suggests that the epitope was not within or near the catalytic region; but the LPL immunoreactivity was completely inhibited in the presence of heparin, which suggests that the MAb reacted with a proteoglycan site of the enzyme. In practice, inhibition by heparin could be eliminated by immunofiltration using the ELIFA system, thus permitting the measurement of both the active and the inactive LPL mass. This MAb has also been used to specifically precipi-

tak polysomcs carryingthe LPL messenger RNA from rat adipose tissue and human blood cells. Altered phospholipid and fatty acid compositions of erythrocyte membranes in familial lipoprotein lipase deficiency CantinE, Brun LD, GagnC C, Murthy MRV, Lupien PJ, Julien P,

12641

QuJbec Lipid Res. Centre, Lava1 Univ. Med. Centre, Ste-Foy, QC, Canada Gl V 4G2

In order to determine the nature of the moderate in vitro hemolysis observed in plasma of patients with familial lipoprotein lipase (LPL) deficiency, we examined the hypo-osmotic fragility and the lipid composition of erythrocytes from these patients. Osmotic fragility was similar in control subjects (n = 21) and patients (n = 26). However, there was a significantly higher concentration of free hemoglobin (0.28 It 0.33 g/l vs. 0.05 i 0.04 g/l in controls, P < 0.005) and of lysophosphatidylcholine (1ysoPc) (12.6 f 5.8% vs. 6.4 rt 1.9% in controls, P < 0.0001) in plasma of LPL-deficient patients. These data, along with a positive correlation between plasma free hemoglobin and IysoPc levels (r = 0.58, P = 0.0001). suggest that this hemolysis is mediated to some extent by the abnormally high concentration of 1ysoPc. Fatty acid levels in the chylomicron-free plasma of 18 patients and 23 control subjects showed that the levels of essential fatty acids (EFA w-3 and o-6 series) were lower (-288, P < 0.001) in LPL deficiency. Moreover, arachidonic acid was lower (P c 0.0005) in erythrocyte membranes from LPL-deficient patients (7.96 f 1.83 mol% vs. 11.28 f 0.94 in controls, Mean f SD). This study suggests that hemolysis in LPL deficiency could result from higher plasma IysoPc and concurrenfly lower erythrocyte arachidonic acid due to a lower supply of dietary fatty acids in the absence of proper chylomicron catabolism by LPL. 12651 m,

Fatty acid composition of adipose tissues in familial lipoprotein lipase deficiency Brun LD, Gagn6 C, Murthy MRV, Lupien PJ, Julien P,

Quebec Lipid Res. Centre, Lava1 Univ. Med. Centre, Ste-Foy, QC, Canada GI V 4G2

Uptake of intravascular esterified fatty acids requires prior hydrolysis of plasma triglycerides through the action of lipoprotein lipase (LPL). The objective of this study was to determine if fat tissue homeostasis in LPL deficiency was maintained by de novo synthesis of fatty acids. Fatty acid composition was studied in biopsies of abdominal and femoral adipose tissues obtained from seven French-Canadian patients with no plasma post-heparin LPL activity and carrying previously identified LPL gene mutations. Nine normolipidemic volunteers were used as controls. Femoral and abdominal tissues of LPL-deficient patients had similar fatty acid profiles, but they showed substantial differences in the content of specific fatty acids from those in controls. In LPL deficiency, the relative concentrations of tissue medium chain fatty acids (ClO-Cl4) were normal, adipose tissue C16:O was elevated (+28%, P
Atherosclerosis X, Montreal, October 1994