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2.P.4 Expression of the human phospholipid transfer protein by the yeast Pichia pastoris
117
Tuesdav 7 October 1997: Posters MOLECULAR
INSIGHTS
I2.
3 Lipid-free apolipoprotein A-I can act as a shuttle to promote cholesterol efflux to apolipopmtein A-I-containing lipoproteios P.G. Frank, Y.L. Marcel. Lipoprotein & Atherosclerosis Group, University of
INTO HDL AND ITS PROPERTIES
El2 P 1
Selective uptake of HDL-CE by human adipose tissue is an LRP-dependent prows Fabienne Benoist, Paulina Lau, Ruth McPherson. University of Ottawa Heart Institute,
Ottawa,
Ottawa
Canada
Since endogenous cholesterol synthesis is limited, adipocytes are dependent on lipoproteins as a cholesterol source. In a previous study, we confirmed previous observations demonstrating selective uptake of HDL-CE by human adipocytes and have identified a novel role for cholesteryl ester transfer protein (CETP) in this process (Benoist et al. Circulation 1996; 94, Suppl I: 464465). In further studies, we have determined the role of the LDL receptor related protein (LRP) in this process. Methods: Fresh adipose tissue fragments were incubated with freshly isolated HDL, labelled with 3H-CE. Incubations were carried out with or without the addition of 39kDA receptor associated protein (RAP) (20 &ml) or a polyclonal antibody against LRP (50 wg/ml). Adipose tissue fragments were also preincubated for 15 min in presence or absence of 100 nM insulin; 3H-CE-HDL was then added and the incubation continued for specific time pWiOdS.
Results: Incubation of adipose tissue with RAP, a receptor antagonist that inhibits ligand interactions with LRP or with an anti-LRP antibody reduced the selective uptake of HDL-CE by human adipose tissue by 16% and 32%, respectively (p < 0.05). It was previously demonstrated that adipocyte exposure to insulin triggers an increase rn the surface presentation of LRP. In our experiments, insulin enhanced by 2 fold the adipocyte uptake of HDL-CE after 30 min of incubation. Following incubation of HDL with adipose tissue, we observed a decrease in HDL-CE concentration in the media and a reciprocal increase in adipocyte plasma membrane cholesterol. Conclusions: These data suggest that the selective uptake of HDL-CE by human adipose tissue is facilitated by insulin-dependent translocation of LRP to me plasma membrane. El2.P 2 The composition and structure of HDL regulate plasma clearance kinetics in rabbits Braschi Svlvie, Tracey Neville, Daniel L. Sparks. Lipoproteins and Atherosclerosis
Group,
Ottawa,
Civic
Hospital,
Ont, KI Y 4E9, Canada
Low apolipoprotein A-I (apo A-I) levels have been primarily associated with increased HDL fractional catabolic rates (FCR). However, the factors that regulate the clearance of HDL from the plasma are unclear. HDL FCR has been shown to be high in patients with low HDL cholesterol levels and appears to be related to changes in the composition and structure of abnormal HDL particles in these patients. In this study, the effect of lipid composition of spherical and discoidal reconstituted HDL particles (LpA-I) on the rates of clearance from rabbit plasma has been investigated. Investigations with well-defined LpA-I have shown that HDL lipid composition affects apoA-I structure and conformation and, in turn, has a significant effect on HDL’s ability to act as substrate for various plasma enzymes (LCAT, hepatic lipase and CETP). We show that LpA-I are also good models for HDL metabolic studies in viva and that particle charge may affect ape A-I clearance. A small spherical Lp2A-I containing phosphatidylcholine and two molecules of apo A-I displayed similar clearance kinetics to exchange labeled HDL. The addition of a cholesteryl ester core or phosphatidylinositol to the Lp2A-I particle increased the negative charge of the particle and led to a significantly slower clearance. Similarly, less negatively charged LpA-I, A-II and discoidal Lp2A-I particles had faster clearance rates than the spherical Lp2A-I. The thermodynamic stability of LpA-1 particles, as analysed by guanidine-HCL denaturation, was also shown to be associated with plasma clearance rates. In general plasma clearance rates were positively and significantly correlated to the surface charge and the thermodynamic stability of LpA-I. In contrast, no relationship was found between clearance rate and particle diameter. We conclude that the HDL composition and structural properties play an important role in HDL plasma metabolism and may regulate the clearance of HDL from plasma.
11th International
Symposium
P
Heart
Institute,
Ottawa,
Canada
Prep-lipoproteins containing apolipoprotein (apo) A-I (LpA-I) have been shown to be the best cholesterol acceptor present in human plasma. However, little is known about the constituents of prep-LpA-I, lipid or protein, which contribute to the specificity of this pathway. Here we show that lipid-free or poor apoA-I which exists in plasma as well as in peripheral fluid, can serve in the first step of the reverse cholesterol transport. ApoA-I recombined with phospholipids to form discoidal LpZA-I mediates an efficient cellular cholesterol efflux from normal human fibroblasts labeled with 3H-cholesterol. In contrast lipid-free apoA-I does not promote any significant cholesterol efflux above that to the control media without any apolipoprotein. However, when the same cells are incubated both lipid-free apoA-I and Lp2A-I, we observe a highly significant enhancement of efflux above that observed with Lp2A-I alone at the same concentration. These results suggest that apoA-I can enhance the ability of Lp2A-I or HDL to promote cholesterol efflux and can act as a catalyst of me first step of the reverse cholesterol transport. This effect of lipid-free apoA-I may be explained by a mechanism similar to what has been attributed to cyclodextrins at low concentration Expression of the human phospholipid transfer protein by the yeast Pichia pastoris V. Guvard-Dannremont’, X.-C. Jiang’, R.W. Milne’ ‘Lipoprotein and
!Tl2.P4
Atherosclerosis Group, University Canada; *Division of Molecular VSA
of Ottawa Heart Institute, Ottawa, Medicine, Columbia University, New York,
Recent studies suggest that the human phospholipid transfer protein (PLTP) may play an important role in the lipoprotein metabolism and may be implicated in reverse cholesterol transport. Little is known, however, of the structure-function relationships of PLTP. In order to identify specific domains of the PLTP implicated in its function we have produced human recombinant PLTP in the methylotrophic yeast, Pichia pastoris, which has been shown to efficiently express and secrete various recombinant mammalian proteins, among them the cholesterol ester transfer protein. A cDNA construct, synthesized by Polymerase Chain Reaction from the full length PLTP cDNA as a template, was designed to allow the convenient cloning of the fragment into the pPICzclA vector (Invitrogen) in frame with the (Yfactor signal sequence (at the 5’ end) and with me sequence coding the myc-epitope and a poly-histidine tag (at the 3’ end). In this construct PLTP is expressed under the control of the alcohol oxidase (AOXI) gene promoter. The pPICZaA vector was then used to target and transform the methylotrophic yeast Pichia pastoris (two strains GS 115 or KIM71 were used). Sequencing of the PLTP cDNA indicated several differences from the published PLTP sequence resulting in two amino acid substitutions. The best results in term of secretion of the recombinant PLTP (rPLTP-myc-his) were obtained with the strain KM71 grown in a minimal medium. After induction, rPLTP-myc-His was detected after 3 days with a maximum a.t4 days of culture. Immunoblot analysis of the medium with monoclonal antibodies against either the myc epitope or the PLTP (5DlO) revealed the presence of an immunoreactive protein with a molecular weight of 78 kDa. By Coomassie blue staining this was the major protein in the medium. In conclusion we have developed an efficient system for expression of human recombinant PLTP. I 2 P.5
1n vi&o factors affecting the concentration of gamma-LpE (y -LpE) in human plasma L. Krimbou, M. Tremblay, H. Jacques,J. Davignon, J.S. Cohn. Hyperlipidemia and Atherosclerosis of Montreal, Quebec, Canada
Research
Group,
Clinical
Research
Inst.
Gamma-LpE (y-LpE), a sphingomyehn-rich lipoprotein which contains apoE as its only protein component, has been proposed to play an important role in cellular cholesterol efflux by acting, like prep I -LpA-I, as an initial acceptor of cell-derived cholesterol. Using two-dimensional non-denaturating polyacrylamide-gradient gel electrophoresis with radiolabeled polyclonal anti-apoE
on Atherosclerosis
Paris,
October
1997
Tuesdav 7 October 1997: Posters MOLECULAR
INSIGHTS
I2.
3 Lipid-free apolipoprotein A-I can act as a shuttle to promote cholesterol efflux to apolipopmtein A-I-containing lipoproteios P.G. Frank, Y.L. Marcel. Lipoprotein & Atherosclerosis Group, University of
INTO HDL AND ITS PROPERTIES
El2 P 1
Selective uptake of HDL-CE by human adipose tissue is an LRP-dependent prows Fabienne Benoist, Paulina Lau, Ruth McPherson. University of Ottawa Heart Institute,
Ottawa,
Ottawa
Canada
Since endogenous cholesterol synthesis is limited, adipocytes are dependent on lipoproteins as a cholesterol source. In a previous study, we confirmed previous observations demonstrating selective uptake of HDL-CE by human adipocytes and have identified a novel role for cholesteryl ester transfer protein (CETP) in this process (Benoist et al. Circulation 1996; 94, Suppl I: 464465). In further studies, we have determined the role of the LDL receptor related protein (LRP) in this process. Methods: Fresh adipose tissue fragments were incubated with freshly isolated HDL, labelled with 3H-CE. Incubations were carried out with or without the addition of 39kDA receptor associated protein (RAP) (20 &ml) or a polyclonal antibody against LRP (50 wg/ml). Adipose tissue fragments were also preincubated for 15 min in presence or absence of 100 nM insulin; 3H-CE-HDL was then added and the incubation continued for specific time pWiOdS.
Results: Incubation of adipose tissue with RAP, a receptor antagonist that inhibits ligand interactions with LRP or with an anti-LRP antibody reduced the selective uptake of HDL-CE by human adipose tissue by 16% and 32%, respectively (p < 0.05). It was previously demonstrated that adipocyte exposure to insulin triggers an increase rn the surface presentation of LRP. In our experiments, insulin enhanced by 2 fold the adipocyte uptake of HDL-CE after 30 min of incubation. Following incubation of HDL with adipose tissue, we observed a decrease in HDL-CE concentration in the media and a reciprocal increase in adipocyte plasma membrane cholesterol. Conclusions: These data suggest that the selective uptake of HDL-CE by human adipose tissue is facilitated by insulin-dependent translocation of LRP to me plasma membrane. El2.P 2 The composition and structure of HDL regulate plasma clearance kinetics in rabbits Braschi Svlvie, Tracey Neville, Daniel L. Sparks. Lipoproteins and Atherosclerosis
Group,
Ottawa,
Civic
Hospital,
Ont, KI Y 4E9, Canada
Low apolipoprotein A-I (apo A-I) levels have been primarily associated with increased HDL fractional catabolic rates (FCR). However, the factors that regulate the clearance of HDL from the plasma are unclear. HDL FCR has been shown to be high in patients with low HDL cholesterol levels and appears to be related to changes in the composition and structure of abnormal HDL particles in these patients. In this study, the effect of lipid composition of spherical and discoidal reconstituted HDL particles (LpA-I) on the rates of clearance from rabbit plasma has been investigated. Investigations with well-defined LpA-I have shown that HDL lipid composition affects apoA-I structure and conformation and, in turn, has a significant effect on HDL’s ability to act as substrate for various plasma enzymes (LCAT, hepatic lipase and CETP). We show that LpA-I are also good models for HDL metabolic studies in viva and that particle charge may affect ape A-I clearance. A small spherical Lp2A-I containing phosphatidylcholine and two molecules of apo A-I displayed similar clearance kinetics to exchange labeled HDL. The addition of a cholesteryl ester core or phosphatidylinositol to the Lp2A-I particle increased the negative charge of the particle and led to a significantly slower clearance. Similarly, less negatively charged LpA-I, A-II and discoidal Lp2A-I particles had faster clearance rates than the spherical Lp2A-I. The thermodynamic stability of LpA-1 particles, as analysed by guanidine-HCL denaturation, was also shown to be associated with plasma clearance rates. In general plasma clearance rates were positively and significantly correlated to the surface charge and the thermodynamic stability of LpA-I. In contrast, no relationship was found between clearance rate and particle diameter. We conclude that the HDL composition and structural properties play an important role in HDL plasma metabolism and may regulate the clearance of HDL from plasma.
11th International
Symposium
P
Heart
Institute,
Ottawa,
Canada
Prep-lipoproteins containing apolipoprotein (apo) A-I (LpA-I) have been shown to be the best cholesterol acceptor present in human plasma. However, little is known about the constituents of prep-LpA-I, lipid or protein, which contribute to the specificity of this pathway. Here we show that lipid-free or poor apoA-I which exists in plasma as well as in peripheral fluid, can serve in the first step of the reverse cholesterol transport. ApoA-I recombined with phospholipids to form discoidal LpZA-I mediates an efficient cellular cholesterol efflux from normal human fibroblasts labeled with 3H-cholesterol. In contrast lipid-free apoA-I does not promote any significant cholesterol efflux above that to the control media without any apolipoprotein. However, when the same cells are incubated both lipid-free apoA-I and Lp2A-I, we observe a highly significant enhancement of efflux above that observed with Lp2A-I alone at the same concentration. These results suggest that apoA-I can enhance the ability of Lp2A-I or HDL to promote cholesterol efflux and can act as a catalyst of me first step of the reverse cholesterol transport. This effect of lipid-free apoA-I may be explained by a mechanism similar to what has been attributed to cyclodextrins at low concentration Expression of the human phospholipid transfer protein by the yeast Pichia pastoris V. Guvard-Dannremont’, X.-C. Jiang’, R.W. Milne’ ‘Lipoprotein and
!Tl2.P4
Atherosclerosis Group, University Canada; *Division of Molecular VSA
of Ottawa Heart Institute, Ottawa, Medicine, Columbia University, New York,
Recent studies suggest that the human phospholipid transfer protein (PLTP) may play an important role in the lipoprotein metabolism and may be implicated in reverse cholesterol transport. Little is known, however, of the structure-function relationships of PLTP. In order to identify specific domains of the PLTP implicated in its function we have produced human recombinant PLTP in the methylotrophic yeast, Pichia pastoris, which has been shown to efficiently express and secrete various recombinant mammalian proteins, among them the cholesterol ester transfer protein. A cDNA construct, synthesized by Polymerase Chain Reaction from the full length PLTP cDNA as a template, was designed to allow the convenient cloning of the fragment into the pPICzclA vector (Invitrogen) in frame with the (Yfactor signal sequence (at the 5’ end) and with me sequence coding the myc-epitope and a poly-histidine tag (at the 3’ end). In this construct PLTP is expressed under the control of the alcohol oxidase (AOXI) gene promoter. The pPICZaA vector was then used to target and transform the methylotrophic yeast Pichia pastoris (two strains GS 115 or KIM71 were used). Sequencing of the PLTP cDNA indicated several differences from the published PLTP sequence resulting in two amino acid substitutions. The best results in term of secretion of the recombinant PLTP (rPLTP-myc-his) were obtained with the strain KM71 grown in a minimal medium. After induction, rPLTP-myc-His was detected after 3 days with a maximum a.t4 days of culture. Immunoblot analysis of the medium with monoclonal antibodies against either the myc epitope or the PLTP (5DlO) revealed the presence of an immunoreactive protein with a molecular weight of 78 kDa. By Coomassie blue staining this was the major protein in the medium. In conclusion we have developed an efficient system for expression of human recombinant PLTP. I 2 P.5
1n vi&o factors affecting the concentration of gamma-LpE (y -LpE) in human plasma L. Krimbou, M. Tremblay, H. Jacques,J. Davignon, J.S. Cohn. Hyperlipidemia and Atherosclerosis of Montreal, Quebec, Canada
Research
Group,
Clinical
Research
Inst.
Gamma-LpE (y-LpE), a sphingomyehn-rich lipoprotein which contains apoE as its only protein component, has been proposed to play an important role in cellular cholesterol efflux by acting, like prep I -LpA-I, as an initial acceptor of cell-derived cholesterol. Using two-dimensional non-denaturating polyacrylamide-gradient gel electrophoresis with radiolabeled polyclonal anti-apoE
on Atherosclerosis
Paris,
October
1997