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4P-1022 Cloning and characterization of cholesteryl ester transfer protein from three shrew
Thursday October 2, 2003: Poster Session Lipid transfer proteins and enzymes reduced the levels of plasma cholesterol and the activities of HMG CoA reductase and CES, while the activities of LPL, LCAT, and CEH were significantly elevated as compared to the untreated high cholesterol fed rats. Conclusion: The results depict that piperine has a modulatory effect on the high cholesterol diet induced alterations on the enzymes of cholesterol and lipoprotein metabolism and also opens up newer avenues on the potential efficacy of piperine in preventing atherogenesis. 4P-1021 ∗
Identification of NPC1 protein as a cholesterol binding protein by photoaffinity labeling
N. Ohgami, S. Sugii, S. Lin, C.C.Y. Chang, T.-Y. Chang. Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA In mammalian cells, the Niemann-Pick Type C1 (NPC1) protein plays an important role in translocating cholesterol and other lipids out of the late endosome via vesicular trafficking. At present, it is not clear whether the NPC1 protein directly binds to cholesterol or not. To test this possibility, we expressed the green fluorescence protein (GFP)-tagged mouse NPC1 protein in mutant CHO cells defective in NPC1 (CT43), then performed intact cell photoaffinity labeling by treating cells with the [3 H]-7,7-azocholestanol/cyclodextrin complex ([3 H]AC/CD, molar ratio = 1:64; Cruz et al., JLR 2002). We then solubilized the cell extracts in the detergent NP-40, and performed immunoprecipitation using anti-GFP monoclonal antibody. The immunoprecipitates underwent Western blotting and radioluminography. The results showed that a single band with the same M.W. as NPC1-GFP (200 kDa) was specifically photolabeled by [3 H]AC/CD. The labeling of NPC1-GFP was completely blocked by including 20-fold excess of unlabeled cholesterol to the labeling mixture. GFP alone was not photolabeled by [3 H]AC/CD. These results suggest that NPC1 is a cholesterol-binding protein. In the future, we will determine the specificity of the ligand for the photolabeling reaction, as well as the nature of the sterol-binding site of the NPC1 protein. 4P-1022
Cloning and characterization of cholesteryl ester transfer protein from tree shrew
W.-w. Zeng, J. Zhang, G. Wu, H. Xue, B.-S. Chen. Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
Atorvastatin effect on paraoxonase and enzymes responsible for HDL remodelling
G. Paragh 1 , L. Illyés 2 , T. Kölbing 1 , M. Harangi 1 , Z. Derdák 1 , A. Kassai 1 , Z. Balogh 1 , G. Pados 3 , L. Seres 1 . 1 Department of Metabolic, First Dept. of Medicine, Medical and Health Science Center, University of Debrecen; 2 Szent Ferenc Hospital, Miskolc; 4 Szent Imre Hospital, Budapest, Hungary Objective: HDL-associated paraoxonase (PON) activity may play an important role in the inhibition of LDL oxidation. Remodelling of HDL by lechitin-cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) is a crucial component of HDL metabolism and function. According to previous studies the effect of atorvastatin on HDL is controversial. Methods: Thirty-three type II.a/II.b hyperlipidemic patients were enrolled in the study. After a washout period, patients received 20mg atorvastatin/day for three months. The serum paraoxonase activity was measured using paraoxon substrate. The serum LCAT was measured spectrophotometrically by cholesterol esterification rate and CETP-mediated transfer was determined by a fluorescence method. OxLDL level was measured using sandwich ELISA. Results: Atorvastatin treatment significantly decreased serum cholesterol (p<0.001), triglyceride (p<0.001) and LDL-C (p<0.001) levels. There was no significant change in the HDL-C level while PON activity (120.43± 84.12; 145.93±102.26 U/l; p<0.001) was significantly increased after atorvastatin treatment. The level of oxidatively modified LDL (oxLDL) was significantly decreased (60.5±15.94; 32.65±9.43 U/l; p<0.001), serum LCAT activity significantly increased (36.78±18.31; 44.75±17.43 mmol/ml/h; p<0.001), while serum CETP activity significantly decreased (151.29±11.35; 143.59±9.4 mmol/ml/h; p<0.001) after atorvastatin therapy. Conclusion: Three months of treatment with atorvastatin favorably effected the lipid profile. It increased LCAT level, decreased CETP level and improved the antioxidant status by increasing serum paraoxonase activity in the studied group of patients. Atorvastatin or its metabolites may reduce oxLDL level through their antioxidant properties. OTKA (T032674), Hungary. 4P-1024
Relationship between human intestinal microsomal triglyceride transfer protein and lipoproteins
G. Tomkin 1,2 , C. Phillips 1 , K. Mullan 1 , D. Owens 2 . 1 Adelaide and Meath Hospital, Dublin; 2 Dept. of Clinical Medicine, Trinity College Dublin, Ireland Microsomal triglyceride transfer protein (MTP) is responsible for the assembly of the chylomicron particle in the intestine and very low density lipoprotein (VLDL) in the liver. In this study we explore the relationship between MTP and lipoproteins. Intestinal biopsies were taken from 18 patients (m/f = 7/11, age 51+10) without renal, hepatic, thyroid disease or diabetes, not on lipid lowering agents who were found to have normal duodenal mucosa following gastroscopy for gastrointestinal symptoms. Ethics committee approval and informed consent were obtained. Within 1 week subjects took an 1100 kcal fat-rich meal, bloods were taken fasting and at 4 and 6h postprandially. Lipoproteins were separated by ultracentrifugation and apo B48 measured by gel electrophoresis. MTP mRNA was measured using the RNase protection assay. Mean MTP mRNA was 11.8 6.2 amol/mg total mRNA. Mean serum cholesterol, triglycerides and HDL were 6.0+0.9, 1.7 0.7 and 1.5+0.6 mmol/l. Chylomicron apo B48 was: fasting 4.0+1.4, 4h 8.1+3.9, and 6h15+11 mg/ml plasma VLDL apo B48 fasting 2.5+1.6, 4h 6.2+4.6, 6h 5.9 4.2. Chylomicron/VLDL apo B48 was double (2.5+2.9vs1.4+0.15) in subjects with MTP above the mean. There was a significant inverse correlation between MTP and serum HDL cholesterol (p<0.007) and MTP and cholesterol/apo B48 (p<0.004). There were significant correlations between MTP and chylomicron apo B48 and phospholipid at 0h p<0.005 and with VLDL apo B48 at 0, and 6h (p<0.025) and 4h (p<0.05). The study suggests that MTP regulates the size of the chylomicron particle. This may have implications for the development of atherosclerosis since small chylomicrons may be more atherogenic. 4P-1025
The recovery of dysfunctional LPL (Asp204-Glu) activity by modification of substrate
T. Murano 1 , K. Shirai 1 , T. Sako 1 , S. Oikawa 2 . 1 Department of Clinical Laboratory Medicine, Sakura General Hospital, Toho University; 2 Department of Third Internal Medicine, Japan Medical University, Japan Purpose: LPL (Asp204-Glu) was found in severe hypertriglyceridemic patient suffered from recurrent pancreatitis. His post heparin plasma LPL mass was almost normal, but the LPL activity was remarkably decreased. The possibilities of the recovery of this enzyme activity were pursued. XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
Objective: To obtain the nucleotide and amino acid sequences of cholesteryl ester transfer protein (CETP) from tree shrew, a good model for its insusceptibility to atherosclerosis. Methods: The cDNA sequence of tree shrew CETP was obtained by utilizing the SMART-RACE technique. Its amino acid sequence was deduced from cDNA sequence and primary and secondary structures were predicted. Results: The sequence of CETP cDNA from tree shrew (accession number in Genbank is AF334033) covers 1636 bp, including 178 bp at 3’ of the untranslated region and a 1458 bp fragment in a coding region, which provides the complete sequence of mature tree shrew CETP, although not the initiator methionine. The first 24bp code for a partial signal peptide. The mature protein consists of 477 amino acids and is longer than that of human by one amino acid (Gly318). Comparing this amino acid sequence with those of other published animals CETP, it reveals that the identity among tree shrew and human, rabbit CETP is 88% and 82% respectively. The protein is extremely hydrophobic as rich of hydrophobic residues, especially at C-terminal, consistent with its function in the transfer of neutral lipids. The amino acid residues that concerned with its function in binding and transfering neutral lipids are highly conserved. There is a deletion of N-linked glycosylation site at Asn342 in tree shrew CETP protein that may be benefit to removing peripheral cholesterol and cholesteryl ester by increasing its activity of transfer cholesteryl ester. A one-nucleotide substitution (G to A, 1379) in tree shrew CETP was detected and resulted an amino acid change, Gln452 in tree shrew CETP instead of Arg451 in human, which effected on the activity of human CETP and the level of total cholesterol. Conclusion: The feature of glycosylation in tree shrew CETP may be involved in the molecular mechanism of its insusceptibility to atherosclerosis.
4P-1023
299
Identification of NPC1 protein as a cholesterol binding protein by photoaffinity labeling
N. Ohgami, S. Sugii, S. Lin, C.C.Y. Chang, T.-Y. Chang. Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA In mammalian cells, the Niemann-Pick Type C1 (NPC1) protein plays an important role in translocating cholesterol and other lipids out of the late endosome via vesicular trafficking. At present, it is not clear whether the NPC1 protein directly binds to cholesterol or not. To test this possibility, we expressed the green fluorescence protein (GFP)-tagged mouse NPC1 protein in mutant CHO cells defective in NPC1 (CT43), then performed intact cell photoaffinity labeling by treating cells with the [3 H]-7,7-azocholestanol/cyclodextrin complex ([3 H]AC/CD, molar ratio = 1:64; Cruz et al., JLR 2002). We then solubilized the cell extracts in the detergent NP-40, and performed immunoprecipitation using anti-GFP monoclonal antibody. The immunoprecipitates underwent Western blotting and radioluminography. The results showed that a single band with the same M.W. as NPC1-GFP (200 kDa) was specifically photolabeled by [3 H]AC/CD. The labeling of NPC1-GFP was completely blocked by including 20-fold excess of unlabeled cholesterol to the labeling mixture. GFP alone was not photolabeled by [3 H]AC/CD. These results suggest that NPC1 is a cholesterol-binding protein. In the future, we will determine the specificity of the ligand for the photolabeling reaction, as well as the nature of the sterol-binding site of the NPC1 protein. 4P-1022
Cloning and characterization of cholesteryl ester transfer protein from tree shrew
W.-w. Zeng, J. Zhang, G. Wu, H. Xue, B.-S. Chen. Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
Atorvastatin effect on paraoxonase and enzymes responsible for HDL remodelling
G. Paragh 1 , L. Illyés 2 , T. Kölbing 1 , M. Harangi 1 , Z. Derdák 1 , A. Kassai 1 , Z. Balogh 1 , G. Pados 3 , L. Seres 1 . 1 Department of Metabolic, First Dept. of Medicine, Medical and Health Science Center, University of Debrecen; 2 Szent Ferenc Hospital, Miskolc; 4 Szent Imre Hospital, Budapest, Hungary Objective: HDL-associated paraoxonase (PON) activity may play an important role in the inhibition of LDL oxidation. Remodelling of HDL by lechitin-cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) is a crucial component of HDL metabolism and function. According to previous studies the effect of atorvastatin on HDL is controversial. Methods: Thirty-three type II.a/II.b hyperlipidemic patients were enrolled in the study. After a washout period, patients received 20mg atorvastatin/day for three months. The serum paraoxonase activity was measured using paraoxon substrate. The serum LCAT was measured spectrophotometrically by cholesterol esterification rate and CETP-mediated transfer was determined by a fluorescence method. OxLDL level was measured using sandwich ELISA. Results: Atorvastatin treatment significantly decreased serum cholesterol (p<0.001), triglyceride (p<0.001) and LDL-C (p<0.001) levels. There was no significant change in the HDL-C level while PON activity (120.43± 84.12; 145.93±102.26 U/l; p<0.001) was significantly increased after atorvastatin treatment. The level of oxidatively modified LDL (oxLDL) was significantly decreased (60.5±15.94; 32.65±9.43 U/l; p<0.001), serum LCAT activity significantly increased (36.78±18.31; 44.75±17.43 mmol/ml/h; p<0.001), while serum CETP activity significantly decreased (151.29±11.35; 143.59±9.4 mmol/ml/h; p<0.001) after atorvastatin therapy. Conclusion: Three months of treatment with atorvastatin favorably effected the lipid profile. It increased LCAT level, decreased CETP level and improved the antioxidant status by increasing serum paraoxonase activity in the studied group of patients. Atorvastatin or its metabolites may reduce oxLDL level through their antioxidant properties. OTKA (T032674), Hungary. 4P-1024
Relationship between human intestinal microsomal triglyceride transfer protein and lipoproteins
G. Tomkin 1,2 , C. Phillips 1 , K. Mullan 1 , D. Owens 2 . 1 Adelaide and Meath Hospital, Dublin; 2 Dept. of Clinical Medicine, Trinity College Dublin, Ireland Microsomal triglyceride transfer protein (MTP) is responsible for the assembly of the chylomicron particle in the intestine and very low density lipoprotein (VLDL) in the liver. In this study we explore the relationship between MTP and lipoproteins. Intestinal biopsies were taken from 18 patients (m/f = 7/11, age 51+10) without renal, hepatic, thyroid disease or diabetes, not on lipid lowering agents who were found to have normal duodenal mucosa following gastroscopy for gastrointestinal symptoms. Ethics committee approval and informed consent were obtained. Within 1 week subjects took an 1100 kcal fat-rich meal, bloods were taken fasting and at 4 and 6h postprandially. Lipoproteins were separated by ultracentrifugation and apo B48 measured by gel electrophoresis. MTP mRNA was measured using the RNase protection assay. Mean MTP mRNA was 11.8 6.2 amol/mg total mRNA. Mean serum cholesterol, triglycerides and HDL were 6.0+0.9, 1.7 0.7 and 1.5+0.6 mmol/l. Chylomicron apo B48 was: fasting 4.0+1.4, 4h 8.1+3.9, and 6h15+11 mg/ml plasma VLDL apo B48 fasting 2.5+1.6, 4h 6.2+4.6, 6h 5.9 4.2. Chylomicron/VLDL apo B48 was double (2.5+2.9vs1.4+0.15) in subjects with MTP above the mean. There was a significant inverse correlation between MTP and serum HDL cholesterol (p<0.007) and MTP and cholesterol/apo B48 (p<0.004). There were significant correlations between MTP and chylomicron apo B48 and phospholipid at 0h p<0.005 and with VLDL apo B48 at 0, and 6h (p<0.025) and 4h (p<0.05). The study suggests that MTP regulates the size of the chylomicron particle. This may have implications for the development of atherosclerosis since small chylomicrons may be more atherogenic. 4P-1025
The recovery of dysfunctional LPL (Asp204-Glu) activity by modification of substrate
T. Murano 1 , K. Shirai 1 , T. Sako 1 , S. Oikawa 2 . 1 Department of Clinical Laboratory Medicine, Sakura General Hospital, Toho University; 2 Department of Third Internal Medicine, Japan Medical University, Japan Purpose: LPL (Asp204-Glu) was found in severe hypertriglyceridemic patient suffered from recurrent pancreatitis. His post heparin plasma LPL mass was almost normal, but the LPL activity was remarkably decreased. The possibilities of the recovery of this enzyme activity were pursued. XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
Objective: To obtain the nucleotide and amino acid sequences of cholesteryl ester transfer protein (CETP) from tree shrew, a good model for its insusceptibility to atherosclerosis. Methods: The cDNA sequence of tree shrew CETP was obtained by utilizing the SMART-RACE technique. Its amino acid sequence was deduced from cDNA sequence and primary and secondary structures were predicted. Results: The sequence of CETP cDNA from tree shrew (accession number in Genbank is AF334033) covers 1636 bp, including 178 bp at 3’ of the untranslated region and a 1458 bp fragment in a coding region, which provides the complete sequence of mature tree shrew CETP, although not the initiator methionine. The first 24bp code for a partial signal peptide. The mature protein consists of 477 amino acids and is longer than that of human by one amino acid (Gly318). Comparing this amino acid sequence with those of other published animals CETP, it reveals that the identity among tree shrew and human, rabbit CETP is 88% and 82% respectively. The protein is extremely hydrophobic as rich of hydrophobic residues, especially at C-terminal, consistent with its function in the transfer of neutral lipids. The amino acid residues that concerned with its function in binding and transfering neutral lipids are highly conserved. There is a deletion of N-linked glycosylation site at Asn342 in tree shrew CETP protein that may be benefit to removing peripheral cholesterol and cholesteryl ester by increasing its activity of transfer cholesteryl ester. A one-nucleotide substitution (G to A, 1379) in tree shrew CETP was detected and resulted an amino acid change, Gln452 in tree shrew CETP instead of Arg451 in human, which effected on the activity of human CETP and the level of total cholesterol. Conclusion: The feature of glycosylation in tree shrew CETP may be involved in the molecular mechanism of its insusceptibility to atherosclerosis.
4P-1023
299