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Transient overexpression of human phospholipid transfer protein (PLTP) induces dynamic changes in the lipoprotein profile of mice
74
7. HDL
Conaluaions: Our data demonstrated that HDL are involved to regulate endothelial homeostasis in hyperlipemias. The inhibition of LDL oxidation could represent one of the underlying mechanism of HDL vascular protection, although fiuther studies will be necessary to lily clarify the benefical effects -of HDL. IPIlo
HYDROLYSIS OF PLATELET-ACTIVATING HUMAN SERUM PARAOXONASE
FACTOR BY
L. Rodrigo’, B. Maolmess’, PN. Durrington’, A. Hemandez’, M.I. Mackness’ . Clinical Research Diuision II, Medicine, Manchester Royal In@marv. Oxford Road, Manchestel; Ml3 9 WL, UK; Departamento de Medicina Legal y Taxicologia, Facultad de Medicina, Uniuersidad de Granada, Avenida de Madrid No II, 18071 Gmnada, Spain
Human serum paraoxonase (PONl) has been shown to be important in the metabolism of phospholipid and cholesteryl-ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein and retarding atherogenesis. -However, the exact substrate specificity of PONl has not been established. In this manwcript we show that purified PONl hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet activating acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not effect the ability of PONl to hydrolyse PAF (30.1f2.8 kmoVmin/mg protein with no inhibitor, 3 1.4f2.2 pmoYmin/mg protein with 100 mh4 inhibitor) or phenyl-acetate (242.6h30.8 vs 240.8~t31.5 pmoYmin/mg protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human HDL but completely abolished the activity of human LDL. Ostrich HDL which does not contain PONl was unable to hydrolyse PAF. This data provides evidenoe that PONl may limit the action of this bioactive, pro-inflammatory phospholipid. IPI11
SECRETION OF HUMAN PARAOXONASE FOR AN HDL ACCEPTOR MECHANISM
IPI12
S. Jaari’, M. Jauhiainen’, K.W. van Dijk*, VM. Olkkonen’, J. Metso’, C. Ehnhohn’ ‘Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 2University of Leiden, Leiden, The Netherlands
PLTP plays an essential role in the metabolism of high density lipoproteins (HDL). Its role in the tisfer of surface remnants from triglyceride-rich particles, VLDL and CM, to HDL during lipolysis is of importance for the maintenance of HDL levels. It also modulates the size and composition of HDL particles, a function of importance in the reverse cholesterol transport process. To investigate the distribution of human PLTP in C57/Bl mice sera adenovirus mediated overexpression of PLTP was used. Size-exclusion chromatography demonstrates the presence of two forms of PLTP in sera, one catalytically active and the other inactive. The PLTP with low specific activity eluted in a similar position as large HDL particles, and the population with high specific activity in the position of small HDL. Similar results have been reported for human subjects. Viral overexpression of PLTP leads to a depletion, of HDL. When the plasma PLTP activity declines, replenishment of HDL occurs. As plasma HDL increasses, transient particle populations, including LDL-sized apoA-IV and apoE-rich particles and small phospholipid containing apoA-II particles, are observed. The increased PLTP activity enhanced the ability of mouse sera to produce pre& HDL (r = 0.95, p < 0.001). The results provide novel evidence that PLTP is an important regulator of HDL and plays a central role in the reverse cholesterol transport.
IPl14
1: EVIDENCE
S.F! Deakin’, I. Leviev’, L. Calabresi*, G. Franceschini*, RW. James’. ‘H6pital Cantonal Unioersitaire de Gendve, Switzerland; 2Uniuersity of Milan, Italy
High density lipoproteins (HDL) are the serum transport vector for the antioxidant enzyme paraoxonase I (PON I). PON I associates with HDL via a hydrophobic signal sequence which is not cleaved before secretion and could allow PON I to attach to cell membranes. We have shown previously that PON I concentrations are decreased in patients with altered HDL states, implying a role for HDL in maintaining serum levels of PON I. The secretion of PON I into serum has not to date been studied. The aim of this study therefore, is to tiestigate the mechanism by which seNm lipoproteins, particularly HDL may influence PON I secretion and serum concentration. CHO cells stably transfected with human PON I were grown in serum free medium or Hanks salt solution in the presence of various serum lipoproteins. Medium PON I concentration and enzyme activity were measured. We show that the presence of HDL is necessary for an efficient and stable secretion of human PON I but that ApoAI alone has only a small positive effect. LDL and VLDL had no positive effect on secretion while protein free phospholipid micelles enhanced secretion but not stability of the enzyme. Using immunofluorescence we localised PON I to the external membrane of transfected cells. The enzyme was found to be active in this cell associated state. Increased PON I secretion induced by HDL was accompanied by a decrease in cell membrane associated enzyme activity. Using the above data we propose a model whereby PON I secretion is enhanced by the presence of an appropriate acceptor which allows the enzyme to be removed from the external membrane. This has implications for the role of HDL in maintaining s&mu PON I concentrations and the consequences of compositional modifications of HDL as occur in dyslipidaemia.
TRANSIENT OVEREXPRESSION OF HUMAN PHOSPHOLIPID TRANSFER PROTEIN IPLTP) INDUCES DYNAMIC CHANGES IN THE LIPOPRhTEIN’PROFILE OF MICE
A NEW ENZYME-LINKED IMMUNOSORBENT ASSAY WITH TWO MONOCLONAL ANTIBODIES TO SPECIFIC EPITOPES MEASURES LECITHIN-CHOLESTEROL ACYLTRANSFERASE IN HUMAN SERUM
K. Kobori’, K. Saito’, S. Ito’, K. Kotani’, K. Ushizawa’, K. Kubono*, T. Ksnno3, M. Manabe’. ‘Diagnostics Research Laboratories, Daiichi Pure Chemicals Co., Ltd. Ryugasaki, Ibamki 301-0852; 2SRL, Inc., Hachioji; 3Hamamatsu University School of medicine, Hamamatsu, Jqan
LCAT catalyzes the formation of cholesteryl esters on HDL by the transfer of fatty acids from the sn-2 position of phosphatidylcholine to cholesterol. LCAT is studied primarily by measurement of its activity, but activity values are known to vary with the substrate, the plasma lipid levels, and the lipoprotein profile. We have developed a new method for precise determination of LCAT protein levels. Hybridoma cells were established by standard techniques. Purified human LCAT and synthetic peptides were used as antigens. The epitope-specificities of the resulting antibodies were determined from their reactivities with synthetic peptides. We then developed an ELISA for measurement of LCAT levels and evaluated correlation between the ELISA data and activity measurement data. We also examined distribution of LCAT in the gel filtrate fraction of serum and accuracy of the ELISA as an assay. Five hybridoma cell lines producing monoclonal antibodies that recognize different epitopes on human LCAT were obtained. These are mouse antihuman LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486 and they react with peptides corresponding to human LCAT amino acid residues Rl59-E179, S258-S273, M273-S294, S352S376, and N415-E440, respectively. Two of the antibodies were used to develop a sandwich ELISA. Monoclonal antibody 36486, which reacts with LCAT C-terminus, is the solid-phase antibody; monoclonal antibody 36487, which reacts with an epitope located in the center of the LCAT primary structure, is the detection antibody. Correlation between our new method and the conventional activity-based measurement of LCAT was confirmed by a significant positive correlation between the liposome substrate method (n = 40, y = 0.013x + 3.1768, r = 0.655) and endogenous self-substrate method (n = 40, y = 0.0543x + 3.8387, r = 6.753). The distribution of LCAT in the gel filtrate fraction of serum was similar to that of the HDL fraction. The validity of this new method was verified with precision (interand intra-assay coefficient of variation < 6.6%) and dilution tests. Conclusion: We have generated five hybridoma cell lines that produce monoclonal antibodies against human LCAT. We used these antibodies to develop a sandwich ELISA that is simple, accurate, and reliable. This new method for measurement of LCAT levels will be useful in the study of the physiological role of LCAT and in clinical study of lipid metabolism.
72nd EAS Congress
7. HDL
Conaluaions: Our data demonstrated that HDL are involved to regulate endothelial homeostasis in hyperlipemias. The inhibition of LDL oxidation could represent one of the underlying mechanism of HDL vascular protection, although fiuther studies will be necessary to lily clarify the benefical effects -of HDL. IPIlo
HYDROLYSIS OF PLATELET-ACTIVATING HUMAN SERUM PARAOXONASE
FACTOR BY
L. Rodrigo’, B. Maolmess’, PN. Durrington’, A. Hemandez’, M.I. Mackness’ . Clinical Research Diuision II, Medicine, Manchester Royal In@marv. Oxford Road, Manchestel; Ml3 9 WL, UK; Departamento de Medicina Legal y Taxicologia, Facultad de Medicina, Uniuersidad de Granada, Avenida de Madrid No II, 18071 Gmnada, Spain
Human serum paraoxonase (PONl) has been shown to be important in the metabolism of phospholipid and cholesteryl-ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein and retarding atherogenesis. -However, the exact substrate specificity of PONl has not been established. In this manwcript we show that purified PONl hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet activating acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not effect the ability of PONl to hydrolyse PAF (30.1f2.8 kmoVmin/mg protein with no inhibitor, 3 1.4f2.2 pmoYmin/mg protein with 100 mh4 inhibitor) or phenyl-acetate (242.6h30.8 vs 240.8~t31.5 pmoYmin/mg protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human HDL but completely abolished the activity of human LDL. Ostrich HDL which does not contain PONl was unable to hydrolyse PAF. This data provides evidenoe that PONl may limit the action of this bioactive, pro-inflammatory phospholipid. IPI11
SECRETION OF HUMAN PARAOXONASE FOR AN HDL ACCEPTOR MECHANISM
IPI12
S. Jaari’, M. Jauhiainen’, K.W. van Dijk*, VM. Olkkonen’, J. Metso’, C. Ehnhohn’ ‘Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 2University of Leiden, Leiden, The Netherlands
PLTP plays an essential role in the metabolism of high density lipoproteins (HDL). Its role in the tisfer of surface remnants from triglyceride-rich particles, VLDL and CM, to HDL during lipolysis is of importance for the maintenance of HDL levels. It also modulates the size and composition of HDL particles, a function of importance in the reverse cholesterol transport process. To investigate the distribution of human PLTP in C57/Bl mice sera adenovirus mediated overexpression of PLTP was used. Size-exclusion chromatography demonstrates the presence of two forms of PLTP in sera, one catalytically active and the other inactive. The PLTP with low specific activity eluted in a similar position as large HDL particles, and the population with high specific activity in the position of small HDL. Similar results have been reported for human subjects. Viral overexpression of PLTP leads to a depletion, of HDL. When the plasma PLTP activity declines, replenishment of HDL occurs. As plasma HDL increasses, transient particle populations, including LDL-sized apoA-IV and apoE-rich particles and small phospholipid containing apoA-II particles, are observed. The increased PLTP activity enhanced the ability of mouse sera to produce pre& HDL (r = 0.95, p < 0.001). The results provide novel evidence that PLTP is an important regulator of HDL and plays a central role in the reverse cholesterol transport.
IPl14
1: EVIDENCE
S.F! Deakin’, I. Leviev’, L. Calabresi*, G. Franceschini*, RW. James’. ‘H6pital Cantonal Unioersitaire de Gendve, Switzerland; 2Uniuersity of Milan, Italy
High density lipoproteins (HDL) are the serum transport vector for the antioxidant enzyme paraoxonase I (PON I). PON I associates with HDL via a hydrophobic signal sequence which is not cleaved before secretion and could allow PON I to attach to cell membranes. We have shown previously that PON I concentrations are decreased in patients with altered HDL states, implying a role for HDL in maintaining serum levels of PON I. The secretion of PON I into serum has not to date been studied. The aim of this study therefore, is to tiestigate the mechanism by which seNm lipoproteins, particularly HDL may influence PON I secretion and serum concentration. CHO cells stably transfected with human PON I were grown in serum free medium or Hanks salt solution in the presence of various serum lipoproteins. Medium PON I concentration and enzyme activity were measured. We show that the presence of HDL is necessary for an efficient and stable secretion of human PON I but that ApoAI alone has only a small positive effect. LDL and VLDL had no positive effect on secretion while protein free phospholipid micelles enhanced secretion but not stability of the enzyme. Using immunofluorescence we localised PON I to the external membrane of transfected cells. The enzyme was found to be active in this cell associated state. Increased PON I secretion induced by HDL was accompanied by a decrease in cell membrane associated enzyme activity. Using the above data we propose a model whereby PON I secretion is enhanced by the presence of an appropriate acceptor which allows the enzyme to be removed from the external membrane. This has implications for the role of HDL in maintaining s&mu PON I concentrations and the consequences of compositional modifications of HDL as occur in dyslipidaemia.
TRANSIENT OVEREXPRESSION OF HUMAN PHOSPHOLIPID TRANSFER PROTEIN IPLTP) INDUCES DYNAMIC CHANGES IN THE LIPOPRhTEIN’PROFILE OF MICE
A NEW ENZYME-LINKED IMMUNOSORBENT ASSAY WITH TWO MONOCLONAL ANTIBODIES TO SPECIFIC EPITOPES MEASURES LECITHIN-CHOLESTEROL ACYLTRANSFERASE IN HUMAN SERUM
K. Kobori’, K. Saito’, S. Ito’, K. Kotani’, K. Ushizawa’, K. Kubono*, T. Ksnno3, M. Manabe’. ‘Diagnostics Research Laboratories, Daiichi Pure Chemicals Co., Ltd. Ryugasaki, Ibamki 301-0852; 2SRL, Inc., Hachioji; 3Hamamatsu University School of medicine, Hamamatsu, Jqan
LCAT catalyzes the formation of cholesteryl esters on HDL by the transfer of fatty acids from the sn-2 position of phosphatidylcholine to cholesterol. LCAT is studied primarily by measurement of its activity, but activity values are known to vary with the substrate, the plasma lipid levels, and the lipoprotein profile. We have developed a new method for precise determination of LCAT protein levels. Hybridoma cells were established by standard techniques. Purified human LCAT and synthetic peptides were used as antigens. The epitope-specificities of the resulting antibodies were determined from their reactivities with synthetic peptides. We then developed an ELISA for measurement of LCAT levels and evaluated correlation between the ELISA data and activity measurement data. We also examined distribution of LCAT in the gel filtrate fraction of serum and accuracy of the ELISA as an assay. Five hybridoma cell lines producing monoclonal antibodies that recognize different epitopes on human LCAT were obtained. These are mouse antihuman LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486 and they react with peptides corresponding to human LCAT amino acid residues Rl59-E179, S258-S273, M273-S294, S352S376, and N415-E440, respectively. Two of the antibodies were used to develop a sandwich ELISA. Monoclonal antibody 36486, which reacts with LCAT C-terminus, is the solid-phase antibody; monoclonal antibody 36487, which reacts with an epitope located in the center of the LCAT primary structure, is the detection antibody. Correlation between our new method and the conventional activity-based measurement of LCAT was confirmed by a significant positive correlation between the liposome substrate method (n = 40, y = 0.013x + 3.1768, r = 0.655) and endogenous self-substrate method (n = 40, y = 0.0543x + 3.8387, r = 6.753). The distribution of LCAT in the gel filtrate fraction of serum was similar to that of the HDL fraction. The validity of this new method was verified with precision (interand intra-assay coefficient of variation < 6.6%) and dilution tests. Conclusion: We have generated five hybridoma cell lines that produce monoclonal antibodies against human LCAT. We used these antibodies to develop a sandwich ELISA that is simple, accurate, and reliable. This new method for measurement of LCAT levels will be useful in the study of the physiological role of LCAT and in clinical study of lipid metabolism.
72nd EAS Congress