Identification of peptides in a phage display library that bind to the apolipoprotein E receptor-2

Identification of peptides in a phage display library that bind to the apolipoprotein E receptor-2

112 Friday, 28 May 1999 Poster presentation: Lipoprotein receptors a valuable new model for the study of hepatic lipoprotein and cholesterol metabol...

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112

Friday, 28 May 1999 Poster presentation: Lipoprotein receptors

a valuable new model for the study of hepatic lipoprotein and cholesterol metabolism. This work was supported by a grant from the British Council/National Research Council of Italy (CNR) Scientific Co-operation Programme.

REGULATION OF CLA-I (CD36 AND LIMP II ANALOGOUS 1) BY ACTIVATORS OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS (PPARS) E Gbaguidi 1 , G. Chinetti 1 , S. Griglio 2, M. Antonucci 2, J.-C. Fruchart 1,

J. Chapman 2, J. Najib 1, B. Staels 1. tlnstitut Pasteur, Lille and 'U 321

References

INSERM, Paris, France

[l] Amicone, L., Spagnoli, F.M., Spath, G., Giordano, S., Tommasini, C., Bernadini, S., DeLuca, V., Della Rocca, C., Weiss, M.C., Comoglio, P.M. and Tripodi, M. (1997) EMBO J. 16, 495-503.

SCAVENGER RECEPTOR B1 (SR-BI) AND CLEARANCE OF apoB-CONTAIN1NG LIPOPROTEINS IN apoE-DEFICIENT MICE N.R. Webb 1, A. Daugherty 1'3, M.C. de Beer 1, M.S. Kindy2, D.R. van der Westhuyzen 1, EC. de Beer TM. Departments of tlnternal Medicine

and "Bmchemwtr 3, and 3the Gill Heart Institute, UniversiO, of Kentuc~, Medical Center, L~vington, Kentuc~' 40536; and 4Department of Veterans Affairs Medical Center. L~rington, Kentuclg., 4051 I, USA The scavenger receptor BI (SR-BI) is a cell surface receptor for high density lipoproteins (HDL). A known function of SR-BI is to mediate the selective uptake of cholesteryl ester from HDL by a non-endocytic mechanism, as occurs in liver and steroidogenic tissues. SR-BI exhibits a broad specificity in its high-affinity binding of low density lipoproteins (LDL), very low density lipoproteins (VLDL), and oxidized LDL, but its role in the metabolism ofapoB-containing lipoproteins is not known. In this study the metabolism of apoB-containing particles by SR-BI was investigated in vitro and in vivo. Two different LDL preparations (human LDL and LDL from LDL receptor deficient mice) were tested as substrates for SRBI in transfected Chinese hamster ovary cells. SR-BI mediated selective uptake of [3H]cholesteryl oleate ether from both LDL types. The effect of adenovirus-mediated overexpression of SR-BI on apoB lipoproteins in vivo was analyzed in three mouse models: apoE deficient, LDL receptor deficient, and human apoB transgenic mice. SR-BI overexpression in livers of LDL receptor deficient and apoB transgenic mice had little effect on plasma LDL levels, whereas plasma HDL levels were markedly decreased. In contrast, SR-BI overexpression in apoE deficient mice resulted in an almost complete loss of VLDL- and LDL-sized remnant fractions as well as HDL. These results demonstrate that SR-BI is able to mediate selective lipid uptake from LDL in vitro, although the receptor has little influence on the levels of typical LDL particles in vivo. SR-BI, however, exerts a marked influence on the metabolism of apoB-containing lipoproteins present in the plasma of apoE -/- mice, indicating a role for this receptor in lipoprotein remnant clearance.

IDENTIFICATION OF PEPTIDES IN A PHAGE DISPLAY LIBRARY THAT BIND TO THE APOLIPOPROTEIN E RECEPTOR-2 X.-M. Sun, A.K. Soutar. Lipoprotein Group, MRC Clinical Sciences

Centre, Imperial College School of Medicine, Hammersmith Hospital, DuCane, Road, London W12 ONN, UK The apolipoprotein E receptor-2 (apoER2) is a member of the LDL receptor (LDLR) gene family whose mRNA is expressed in brain and placenta as several splicing variants. Little is known of the physiological function of apoER2, although its cytoplasmic domain contains novel potential binding motifs for signalling proteins. Cells transfected with apoER2 variants express the predicted proteins, as detected by immunoblotting with a specific antipeptide antiserum raised against the first Cys-rich repeat and by binding of receptor associated protein (RAP), and all bind apoE-containing betaVLDL, despite lacking Cys-rich repeats that are essential in the LDLR for ligand binding. However, no degradation of lipoprotein occurred, suggesting that the mechanism of interaction of apoE with apoER2 differs from that with the LDLR. To investigate other possible protein ligands for apoER2, we have expressed the putative ligand binding domain and screened the expressed protein with a random 26-mer peptide phage display library, kindly provided by Dr M. Doyle, Chiron Corporation. After three rounds of panning, 82 individual phage inserts were sequenced; 16 different predicted peptides were found, three strongly enriched (26, 21 and 13/82). ELISA assays showed that all three bound with high affinity. These peptides may identify new ligands for apoER2 or potential antagonists that will be useful for defining its physiological function.

Regulation of expression of scavenger receptors, a class of multiligand cell surface receptors for modified lipoproteins, is thought to play a critical role in the accumulation of lipids by macrophages in atherosclerosis. Human CLA-I, the homolog of murine scavenger receptor B type I(SRBI), binds HDL with high affinity and is involved in the reverse transport of cholesterol. Peroxisome proliferator activated receptor (PPAR) are ligand dependent transcription factors which, upon heterodimerization with the 9-cis retinoic acid receptor (RXR), regulate the expression of target genes involved in lipids metabolism. Given the role of PPARs in monocyte differentiation. macrophage apoptosis and inflammatory control, we investigated the regulation of CLA-I expression by PPAR activators. Western blot analysis using a specific CLA-I antibody, demonstrated that PPARa (Wy 14,643) and PPARg (BRL49653) ligands induced CLA-I protein expression in isolated human monocytes. This effect was enhanced in the presence of LGI069, an RXR specific synthetic ligand and also found in human differentiated macrophages. By immunohistochemical methods, we showed that SRB-I protein expression was enhanced in aortic atherosclerotic lesions ofApo E knock out mice treated with troglitazone (a PPARg ligand) compared to control animals Taken together, these data provide a novel function of PPARs in the control of macrophage lipid content, with likely consequences in atherosclerosis development. PHYLOGENESIS AND DEVELOPING OF FATTY ACIDS TRANSPORT INTO CELLS V.N. Titov. National CardioloKv Research Centre, Mosco,; Russia We suppose that in phylogenesis the formation of fatty acids transport into cells has occurred for four stages which form successively apolipoproteins (apo) A, B (B-48 and B-100) and E. Lipoproteins (LP) of high and Io~v density carry separately the polyenic and saturated acids to cells. At the first stage apoA-LP3, transport fatty acids as polar lipids: polyenic - in phospholipids, saturated - in mono- and diacylglycerides. At the second stage apoA48s conduct a passive transfer of saturated fatty acids as nonpolar triacylglycerides: VLDL albumin cell; polyenic fatty acids as nonpolar cholesteryl esters by means of liquid endocytosis. At the third stage isoprotein apoB100 creates an active transport to cell of polyenic acids as cholesteryl esters through apoB-100 endocytosis of low density LP. At the fourth stage apoE forms an active transport of saturated as well as polyenic acids. ApoE forms a cooperative ligand with each of previous apoproteins: apoE/B-48, apoE/B-100 and apoE/A-I; cells synthesize receptors. ApoE/B-48 receptor delivers exogenic saturated acids as triacylglicerides of chylomicrons into hepatocytes. ApoE/B-100 endocytosis transports endogenic saturated acids as triacylglicerides of very low density LP into cells. ApoE/A-I receptor creates transport into cells of polyenic acids as cholesteryl esters of very low density LP; the transport alternative one to that of apoB/100 and LDL. ApoE/A-I endocytosis of polyenic acids has predetermined the resistance to atherosclerosis. ApoB-100 endocytosis of polyenic acids has determined the sensitivity to atherosclerosis. VLDL ACTIVATION OF PLASMINOGEN ACTIVATOR INHIBITOR1 (PAl-I) EXPRESSION: INVOLVEMENT OF THE VLDL RECEPTOR L. Nilsson, M. G/tfvels, L. Musakka, K. Ensler, D.K. Strickland, B. Angelin, A. Hamsten, P. Eriksson. Karolinska Institute, King Gustaf V

Research Institute, Karolinska Hospital, Stockholm, Sweden The potential role of the very low density lipoprotein (VLDL) receptor in mediating VLDL-induced plasminogen activator inhibitor-I (PAl-I) expression in vitro was studied. Cultured endothelial cells incubated with VLDL showed an increase secretion of PAI-I. This response to VLDL could be completely prevented by receptor associated protein (RAP) and partially blocked by rabbit polyclonal anti-VLDL receptor IgG. Furthermore, Chinese hamster ovary (CHO) control cells overexpressing the VLDL receptor were transiently transfected with a PAI-I promoter-reporter construct and incubated with VLDL. The PAl-l promoter activity in response to VLDL

71st EAS Congress and Satellite Symposia