[show abstract][hide abstract] ABSTRACT: Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE(-/-) and LDLr(-/-) mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE(-/-) (p<0.05, n = 11) and LDLr(-/-) mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr(-/-) strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.
PLoS ONE 01/2012; 7(12):e52634. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The usefulness of conjugated linoleic acid (CLA) as a nutraceutical remains ambiguous. Our objective was, therefore, to investigate the effect of CLA on body composition, blood lipids, and safety biomarkers in overweight, hyperlipidemic men. A double-blinded, 3-phase crossover trial was conducted in overweight (BMI ≥ 25 kg/m(2)), borderline hypercholesterolemic [LDL-cholesterol (C) ≥ 2.5 mmol/L] men aged 18-60 y. During three 8-wk phases, each separated by a 4-wk washout period, 27 participants consumed under supervision in random order 3.5 g/d of safflower oil (control), a 50:50 mixture of trans 10, cis 12 and cis 9, trans 11 (c9, t11) CLA:Clarinol G-80, and c9, t11 isomer:c9, t11 CLA. At baseline and endpoint of each phase, body weight, body fat mass, and lean body mass were measured by DXA. Blood lipid profiles and safety biomarkers, including insulin sensitivity, blood concentrations of adiponectin, and inflammatory (high sensitive-C-reactive protein, TNFα, and IL-6) and oxidative (oxidized-LDL) molecules, were measured. The effect of CLA consumption on fatty acid oxidation was also assessed. Compared with the control treatment, the CLA treatments did not affect changes in body weight, body composition, or blood lipids. In addition, CLA did not affect the β-oxidation rate of fatty acids or induce significant alterations in the safety markers tested. In conclusion, although no detrimental effects were caused by supplementation, these results do not confirm a role for CLA in either body weight or blood lipid regulation in humans.
Journal of Nutrition 07/2011; 141(7):1286-91. · 4.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.
The Journal of Lipid Research 03/2011; 52(6):1170-80. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evidence suggests that minor isomers of conjugated linoleic acid (CLA), such as trans8, cis10 CLA, can elicit unique biological effects of their own. In order to determine the effect of a mixture of t8, c10+c9, t11 CLA isomers on selected aspects of lipid metabolism, 3T3-L1 preadipocytes were differentiated for 8 days in the presence of 100 microM linoleic acid (LA); t8, c10+c9, t11 CLA; t10, c12+c9, t11 CLA or purified c9, t11 CLA. Whereas supplementation with c9, t11 and t10, c12+c9, t11 CLA resulted in cellular triglyceride (TG) concentrations of 3.4 +/- 0.26 and 1.3 +/- 0.11 microg TG/microg protein, respectively (P < 0.05), TG accumulation following treatment with CLA mixture t8, c10+c9, t11 was significantly intermediate (2.5 +/- 0.22 microg TG/microg protein, P < 0.05) between the two other CLA treatments. However, these effects were not attributable to an alteration of the Delta(9) desaturation index. Adiponectin content of adipocytes treated with t8, c10+c9, t11 mixture was similar to the individual isomer c9, t11 CLA, and both the t8, c10+c9, t11 and c9, t11 CLA groups were greater (P < 0.05) than in the t10, c12+c9, t11 CLA group. Overall, these results suggest that t8, c10+c9, t11 CLA mixture affects TG accumulation in 3T3-L1 cells differently from the c9, t11 and t10, c12 isomers. Furthermore, the reductions in TG accumulation occur without adversely affecting the adiponectin content of these cells.
[show abstract][hide abstract] ABSTRACT: Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein. During lipoprotein assembly, it recruits phospholipids and triacylglycerols (TAG) into TAG-rich lipoprotein particles. It remains bound to secreted lipoproteins during lipid metabolism in plasma. The beta1 region (residues 827-1880) of apoB has a high amphipathic beta strand (AbetaS) content and is proposed to be one region anchoring apoB to lipoproteins. The AbetaS-rich region between apoB37 and apoB41 (residues 1694-1880) was cloned, expressed, and purified. The interfacial properties were studied at the triolein/water (TO/W) and air/water (A/W) interfaces. ApoB[37-41] is surface-active and adsorbs to the TO/W interface. After adsorption the unbound apoB[37-41] was removed from the aqueous phase. Adsorbed apoB[37-41] did not desorb and could not be forced off by increasing the surface pressure up to 23 mN/m. ApoB[37-41] adsorbed on the TO/W interface was completely elastic when compressed and expanded by +/-13% of its area. On an A/W interface, the apoB[37-41] monolayer became solid when compressed to 4 mN/m pressure indicating extended beta-sheet formation. It could be reversibly compressed and expanded between low pressure and its collapse pressure (35 mN/m). Our studies confirm that the AbetaS structure of apoB[37-41] is a lipid-binding motif that can irreversibly anchor apoB to lipoproteins.
The Journal of Lipid Research 03/2009; 50(7):1340-52. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conjugated linoleic acids (CLA) are isomeric forms of linoleic acid (LA) containing two conjugated sites of unsaturation. The most abundant dietary form of CLA is the cis-9,trans-11 (c-9,t-11) isomer that is found in the fatty tissues and milk of ruminant animals. CLA can also be acquired by ingestion of supplements, which are usually equimolar mixtures of the c-9,t-11 and t-10,c-12 CLA. For more than a decade, the potential for CLA to modify atherosclerosis in animal models has been examined. However, to date, the studies have failed to reach consensus on whether CLA can be effective in reducing the incidence or severity of atherosclerotic lesions, or whether or not plasma lipid and lipoprotein levels can be improved with CLA supplementation. This review will examine the evidence for and against a role for CLA in atherosclerosis, with a focus on the rabbit, the hamster, and the apoE-deficient mouse.
Biochemistry and Cell Biology 09/2008; 86(4):293-301. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.
The Journal of Lipid Research 07/2008; 49(10):2149-60. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans-10, cis-12 conjugated linoleic acid (t-10, c-12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t-10, c-12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 microM t-10, c-12 CLA with or without 10 microM troglitazone, a PPARgamma agonist, or 1 microM GW9662, a PPARgamma antagonist, and adiponectin and TG levels were analyzed. Treatment with t-10, c-12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARgamma mass. Troglitazone was able to reverse the t-10, c-12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARgamma agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t-10, c-12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARgamma-dependent and PPARgamma-independent mechanisms.
The Journal of Lipid Research 04/2008; 49(3):550-62. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dietary supplementation with conjugated linoleic acid (CLA) has been shown, in several animal models, to decrease the development of atherosclerosis. The mechanism behind the anti-atherogenic properties of CLA is not clear. The objectives of this study were to determine the effect of CLA on atherosclerosis, lipoprotein and liver lipid metabolism, and plasma adiponectin and insulin in apoE(-/-) mice fed an atherogenic (16%, w/w fat; 1.25%, w/w cholesterol) diet. Mice were fed the diet with or without supplementation of linoleic acid (LA), c-9,t-11 CLA, t-10,c-12 CLA, or a 1:1 mixture of the two CLA isomers, at a concentration of 0.5% (w/w), for 12 weeks. Relative to the LA group, CLA supplementation had no significant effect on the lesion area in either en face preparations of the aorta or in aortic root cross-sections. Plasma triacylglycerol and cholesterol concentrations were higher in the t-10,c-12 CLA group than all other treatment groups and liver weight was also increased in this group due to a three-fold increase in liver triacylglycerol. Supplementation with t-10,c-12 CLA or mixed CLA reduced plasma adiponectin levels, whereas t-10,c-12 CLA increased plasma insulin levels. Liver triglycerides correlated directly with blood glucose and plasma insulin and inversely with plasma adiponectin. We conclude that dietary supplementation with CLA does not affect atherosclerosis of the apoE(-/-) mouse on a high-cholesterol diet. Furthermore, t-10,c-12 CLA causes adverse changes in adipocyte function and plasma and liver lipid metabolism, which are partially ameliorated by the inclusion of the c-9,t-11 CLA isomer.
[show abstract][hide abstract] ABSTRACT: Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.
Journal of Biological Chemistry 09/2007; 282(33):24270-83. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine whether adipocyte enhancer binding protein (AEBP) 1, a transcriptional repressor that is down-regulated during adipogenesis, functions as a critical regulator of adipose tissue homeostasis through modulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) tumor suppressor activity and mitogen-activated protein kinase (MAPK) activation.
We examined whether AEBP1 physically interacts with PTEN in 3T3-L1 cells by coimmunoprecipitation analysis. We generated AEBP1-null mice and examined the physiological role of AEBP1 as a key modulator of in vivo adiposity. Using adipose tissue from wild-type and AEBP1-null animals, we examined whether AEBP1 affects PTEN protein level.
AEBP1 interacts with PTEN, and deficiency of AEBP1 increases adipose tissue PTEN mass. AEBP1-null mice have reduced adipose tissue mass and enhanced apoptosis with suppressed survival signal. Primary pre-adipocytes from AEBP1-null adipose tissues exhibit lower basal MAPK activity with defective proliferative potential. AEBP1-null mice are also resistant to diet-induced obesity, suggesting a regulatory role for AEBP1 in energy homeostasis.
Our results suggest that AEBP1 negatively regulates adipose tissue PTEN levels, in conjunction with its role in proliferation and differentiation of pre-adipocytes, as a key functional role in modulation of in vivo adiposity.
[show abstract][hide abstract] ABSTRACT: In McA-RH7777 cells stably expressing human apolipoprotein (apo) B100, treatment with oleic acid (18:1(n-9)) promoted whereas treatment with eicosapentaenoic acid (EPA, 20:5(n-3)) attenuated assembly and secretion of VLDL. Under conditions where the cells were cultured in the presence of 20% serum, EPA (0.4 mM) had marginal effect on the secretion of total apoB100 (determined by pulse-chase analysis) but decreased (by 50%) secretion of triacylglycerol (TG), indicating that the inhibitory effect of EPA was exerted primarily on TG-rich VLDL. Analysis of phospholipid mass and species by tandem mass spectrometry showed increased phosphatidylethanolamine (PE) in EPA-treated cells, the increase was significant in the distal Golgi membranes (by 170%) and endoplasmic reticulum (by 116%). Lipid pulse-chase studies showed a major distinction between phospholipid species containing 20:5(n-3) and 18:1(n-9), which in turn was associated with distinct compartmentalization of TG containing 20:5(n-3) or 18:1(n-9) between cytosol and microsomes and their recruitment during VLDL assembly. Thus, 18:1-TG was secreted as VLDL but 20:5-TG was not. These results suggest that EPA attenuation of VLDL secretion is associated with impaired utilization of TG derived from phospholipid remodeling.
Biochimica et Biophysica Acta 05/2006; 1761(4):463-73. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (LA, C18:2 cis-9, cis-12) that are reported to have important biological activities, including protection against atherosclerosis. In this study, the potential role of the individual cis-9, trans-11 and trans-10, cis-12 isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster. Supplementation of a high-fat, high-cholesterol diet (HFHC) with 1% (w/w) cis-9, trans-11 CLA or trans-10, cis-12 CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1% (w/w) LA. Very low density lipoprotein cholesterol (VLDL-C) was lower and plasma triglycerides (TG) were higher in diets where C18:2 fatty acid was added to the HFHC diet, but neither the cis-9, trans-11 CLA group nor trans-10, cis-12 CLA group was significantly different from the LA control group. CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C). Trans-10, cis-12 CLA increased high density lipoprotein cholesterol (HDL-C) levels compared to LA or cis-9, trans-11 CLA (P<0.02), and although the ratio of non-HDL-C:HDL-C in the cis-9, trans-11 CLA group (1.11+/-0.54) and the trans-10, cis-12 CLA group (1.11+/-0.21) was lower than the LA group (1.29+/-0.45), the reduction did not reach statistical significance. Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal lesions. Compared to the LA group (60+/-11%), both the cis-9, trans-11 CLA group (38+/-8%) and the trans-10, cis-12 CLA group (28+/-7%) had fewer sections displaying a fatty streak lesion, although the differences did not reach statistical significance. These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster, but when compared to LA, the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.
Biochimica et Biophysica Acta 07/2005; 1734(3):269-76. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Treatment of epilepsy or bipolar disorder with valproic acid (VPA) induces weight gain and increased serum levels for the satiety hormone, leptin, through an unidentified mechanism. In this study we tested the effects of VPA, a short-chain branched fatty acid (C8:0), on leptin biology and fatty acid metabolism in 3T3-L1 adipocytes. VPA significantly reduced leptin secretion in a dose-dependent manner. Because fatty acid accumulation has been hypothesized to block leptin secretion, we tested the effect of VPA on fatty acid metabolism. Using 14C-radiolabeled VPA, we found that the 14C was mainly incorporated into triacylglycerol. VPA did not alter lipogenesis from acetate, nor did it change the amount of intracellular free fatty acids available for triacylglycerol synthesis. Decreased leptin secretion was accompanied by a reduction in leptin mRNA, even though VPA treatment did not alter the protein levels for known transcription factors affecting leptin transcription including: CCAAT/enhancer binding protein-alpha, peroxisome proliferator-activated receptor-gamma, or steroid regulatory element binding protein 1a. VPA altered levels of leptin mRNA independent of de novo protein synthesis without affecting leptin mRNA degradation. This report demonstrates that VPA decreases leptin secretion and mRNA levels in adipocytes in vitro, suggesting that VPA therapy may be associated with altered leptin homeostasis contributing to weight gain in vivo.
[show abstract][hide abstract] ABSTRACT: Conjugated linoleic acids (CLAs) are isomeric forms of the 18:2 fatty acid that contain conjugated sites of unsaturation. Although CLAs are minor components of the diet, they have many reported biological activities. For nearly a decade, the potential for CLA to modify the atherosclerotic process has been examined in animal models, and studies of supplementation of the human diet with CLA were started with the anticipation that such an intervention could also reduce the risk of cardiovascular disease. Central to the hypothesis is the expectation that dietary modification could alter plasma lipid and lipoprotein metabolism toward a more cardioprotective profile. This review examines the evidence in support of the hypothesis and the mechanistic studies that lend support for a role of CLA in hepatic lipid and lipoprotein metabolism. Although there are still limited studies in strong support of a role for CLA in the reduction of early atherosclerotic lesions, there has been considerable progress in defining the mechanisms of CLA action. CLA could primarily modulate the metabolism of fatty acids in the liver. The tools are now available to examine isomer-specific effects of CLA on hepatic lipid and lipoprotein metabolism and the potential of CLA to modify hepatic gene expression patterns. Additional animal and cell culture studies will increase our understanding of these unusual fatty acids and their potential for health benefits in humans.
American Journal of Clinical Nutrition 07/2004; 79(6 Suppl):1169S-1174S. · 6.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apolipoprotein B (apoB)-48 contains a region termed the beta1 domain that is predicted to be composed of extensive amphipathic beta-strands. Analysis of truncated apoB variants revealed that sequences between the carboxyl termini of apoB-37 and apoB-42 governed the secretion efficiency and intracellular stability of apoB. Although apoB-37, apoB-34, and apoB-29 were stable and secreted efficiently, apoB-42 and apoB-100 were secreted poorly and were degraded by an acetyl-leucyl-leucyl-norleucinal (ALLN)-sensitive pathway. Amino acid sequence analysis suggested that a segment between the carboxyl termini of apoB-38 and apoB-42 was 63% homologous to fatty acid binding proteins (FABPs), which contain orthogonal beta-sheets. To test the hypothesis that sequences from the beta1 domain are involved in apoB degradation, fusion proteins were created that contained apoB-29 linked to fragments derived from the beta1 domain of apoB or to liver FABP. Fusion proteins containing the beta1 domain segments apoB-34-42 or apoB-37-42 were degraded rapidly, whereas other fusion proteins were stable and secreted efficiently. Degradation was ALLN-sensitive, and the apoB-34-42 segment increased the association of the apoB protein with the cytosolic surface of the microsomal membrane. Our data suggest that the presence of specific sequences in the beta1 domain of human apoB increases degradation by promoting the cytosolic exposure of the protein, although not all regions of the beta1 domain are functionally equivalent.
The Journal of Lipid Research 03/2004; 45(2):366-77. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.
Biochimica et Biophysica Acta 04/2003; 1646(1-2):164-72. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via α-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the β-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure–function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1–5) of human LCAT. Western blot analysis showed that the mutant proteins (Δ1–Δ5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Δ1 mutant) resulted in a dramatic loss of α-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full β-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Δ2 mutant) abolished α-LCAT activity and reduced β-LCAT activity to 12% of wild type. Nevertheless, LCATΔ1 and Δ2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the α-LCAT cofactor apoA-I (in Δ1) and/or loss of enzyme activity (in Δ1–Δ5). Since the Δ1 and Δ2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.
Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics. 01/2003;
[show abstract][hide abstract] ABSTRACT: We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N(158)) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N(158), N(956), N(1341), N(1350), and N(1496)), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.
The Journal of Lipid Research 09/2002; 43(9):1496-507. · 4.39 Impact Factor