Early-glycation of apolipoprotein E: effect on its binding to LDL receptor, scavenger receptor A and heparan sulfates Isabelle Laffont a , Vladimir V. Shuvaev a , Olivier Briand b , Sophie Lestavel b , Anne Barbier a , Naoyuki Taniguchi c , Jean-Charles Fruchart b , Ve ´ronique Clavey b , Ge ´rard Siest a, * a INSERM U525 (Equipe 4), Centre du Me ´dicament, Universite ´ Henri Poincare ´ Nancy 1, 30, rue Lionnois, 54000 Nancy, France b INSERM U545, Institut Pasteur de Lille, Universite ´ Lille 2, 1, rue du Professeur Calmette, 59019 Lille, France c Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Received 6 September 2001; received in revised form 5 February 2002; accepted 20 February 2002 Abstract Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line 125 I-LDL, and on a murine macrophage cell line (J774) 125 I-acetylated LDL, and unlabeled apoE/ phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Apolipoprotein E; Glycation; LDL receptor; Scavenger receptor A; Heparan sulfate 1. Introduction Atherosclerosis and its complications are the major cause of death among diabetic patients [1,2]. A large number of lipid abnormalities has been described in diabetic subjects who are under poor glycaemic control and includes increased levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), and decreased levels of high density lipoprotein (HDL) [3–5]. Glycation is the result of the irreversible attachment of sugar moieties to NH 2 -protein groups, and is believed to play an important role in cardiovascular disease development among diabetic population through an impairment of lipoprotein functions. Glycation of LDL, HDL and VLDL in diabetes mellitus has already been reported [6]. Studies of the functional con- sequences of LDL glycation report that LDL once glycated are poorly recognized by the LDL receptor (LDL-R) and display higher susceptibility to oxidation reaction. Modified LDL accumulate in plasma and can be taken up by macro- phages through an interaction with scavenger receptors, thus contributing to foam cell formation, characteristic features of atherosclerotic plaques [7–9]. Glycation of VLDL and HDL also affects their functional activities: glycated VLDL display a higher residence time in plasma, are poorer sub- strates for lipoprotein lipase than non-glycated VLDL [10], and glycation impairs paraoxonase activity in HDL [11]. It has been demonstrated that apolipoproteins (apo) A-I, A-II, B, C-I and E are glycated in diabetic subjects [12]. Moreover, the glycation process has been shown to impair their func- tions: the formation of advanced glycation end products (AGE) in apoB has been demonstrated to be involved in the loss of recognition of LDL by the LDL-R [13,14]. It has also been demonstrated that glycation inhibits the lipid 1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1388-1981(02)00188-9 * Corresponding author. Tel.: +33-3-83-17-88-34; fax: +33-3-83-32- 13-22. E-mail address: Gerard.Siest@ctrmed.u-nancy.fr (G. Siest). www.bba-direct.com Biochimica et Biophysica Acta 1583 (2002) 99 – 107