Nathalie Theret

Structural domain of apolipoprotein A-I involved in its interaction with cells

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Comparison between fat intake assessed by a 3-day food record and phospholipid fatty acid composition of red blood cells: Results from the monitoring of cardiovascular disease-Lille study

We investigated the relationship between assessment of fatty acid intake by a 3-day food record and by capillary gas chromatography of erythrocyte phospholipid fatty acid. The study was performed in a sample of 244 men aged 45 to 66 years from the general population who were participating in the Monitoring of Cardiovascular Disease (MONICA)-Lille survey. The relationship between each nutrient and food item and erythrocyte phospholipid fatty acid was investigated by a regression model on proportion including each food item and nutrient as a dependent variable and percentage of fatty acid and covariables (nonalcoholic energy intake, age, alcohol intake, and smoking) as independent variables. Polyunsaturated fat and linoleic acid intake were positively correlated with linoleic acid content of erythrocytes (beta = 0.641 and 0.604, respectively, P < .001). Monounsaturated and saturated fat intake were correlated with oleic acid (beta = 0.375 and 0.373, respectively, P < .01). Fish intake correlated positively with docosahexaenoic acid (DHA) (beta = 0.383, P < .001) and negatively with arachidonic acid (beta = -0.509, P < .01). These data confirm, on a group level, a good relationship between assessment of polyunsaturated fat intake by a 3-day record and linoleic acid content of erythrocyte membranes. These data suggest that erythrocyte oleic acid content is a marker of both saturated and monounsaturated fat intake.

The relationship between the phospholipid fatty acid composition of red blood cells, plasma lipids, and apolipoproteins

This study examined the relationship between the fatty acid composition of red blood cell phospholipids and lipid markers of atherosclerotic risk in an urban male population aged 45 to 66 years. There was a surprisingly significant positive association between the docosahexaenoic acid ([DHA] 22:6n-3) content of erythrocyte phospholipids and the following risk markers: plasma cholesterol (P < .01), low-density lipoprotein (LDL) cholesterol (P < .01), apolipoprotein (apo) B (P < .05), and apo B-containing lipoprotein particles (P < .05) recognized by a monoclonal antibody (LpBL3). On the other hand, phospholipid alpha-linolenate was positively correlated with apo A-I and high-density lipoprotein (HDL) cholesterol levels (P < .05), while arachidonate showed an inverse relationship with plasma cholesterol (P < .05). There was a negative association between palmitoleic acid and apo B (P < .01) and LpBL3 (P < .001); the latter showed a negative association with stearic acid (P < .001). These interesting findings emphasize the beneficial effect on atherosclerotic risk markers of dietary n-6 polyunsaturated and monounsaturated fatty acids, and suggest that long-chain n-3 polyunsaturated fatty acids (DHA) could have an adverse effect on some of the lipid risk markers.

Lipoproteins containing apolipoprotein A-IV: Composition and relation to cholesterol esterification

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Apolipoprotein A-I-Containing Particles and Reverse Cholesterol Transport in IDDM

Lipoprotein particles containing apoA-l but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.

Tangier disease : isolation and characterization of LpA-I, LpA-II, LpA-I:A-II and LpA-IV particles from plasma

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 +/- 0.14 and 0.14 +/- 0.04 mumol of cholesterol esterified/h/micrograms of protein, and 7 +/- 2.5 and 1.4 +/- 0.3 mumol of cholesteryl ester transferred/h/micrograms of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2-1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 +/- 0.16 and 0.05 +/- 0.01 mumol of cholesterol esterified/h/micrograms of protein and, 41 +/- 3.7 and 1 +/- 0.4 mumol of cholesteryl ester transferred/h/micrograms of protein). The LpA-II particle in TD represented in fact an LpA-II:A-IV complex (75% mol apoA-II and 22% mol apoA-IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein A-I Containing Particles

Many epidemiological studies have indicated that the plasma level of high density lipoproteins (HDL) is inversely correlated with the risk for coronary artery disease1. It has been hypothesized that HDL exerts this protective effect by the “reverse” transport of excess cholesterol from peripheral tissues to the liver2.

Acylgalactosylceramides in Developing Dysmyelinating Mutant Mice

Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which co‐nigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography‐mass spectrometry analysis of both ester‐ and amide‐linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.

Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance

This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).

Intramyelinic conversion of cerebrosides into acylgalactosylceramides

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [3H]palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [14C]palmitic acid, 2mM ATP, 50 μM CoA and 10 mM MgCl2 and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [3H] cerebrosides and [14C]palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37°C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80°C. Omission of ATP, CoA, MgCl2 or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.