title: The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase.
abstract: OBJECTIVE
  Hyperglycaemia, one of the main features of diabetes, results in non-enzymatic glycation of plasma proteins, including apolipoprotein A-I (apoA-I), the most abundant apolipoprotein in HDL. The aim of this study was to determine how glycation affects the structure of apoA-I and its ability to activate lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport.
  
  
  METHODS
  Discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and apoA-I ([A-I]rHDL) were prepared by the cholate dialysis method and glycated by incubation with methylglyoxal. Glycation of apoA-I was quantified as the reduction in detectable arginine, lysine and tryptophan residues. Methylglyoxal-AGE adduct formation in apoA-I was assessed by immunoblotting. (A-I)rHDL size and surface charge were determined by non-denaturing gradient gel electrophoresis and agarose gel electrophoresis, respectively. The kinetics of the LCAT reaction was investigated by incubating varying concentrations of discoidal (A-I)rHDL with a constant amount of purified enzyme. The conformation of apoA-I was assessed by surface plasmon resonance.
  
  
  RESULTS
  Methylglyoxal-mediated modifications of the arginine, lysine and tryptophan residues in lipid-free and lipid-associated apoA-I were time- and concentration-dependent. These modifications altered the conformation of apoA-I in regions critical for LCAT activation and lipid binding. They also decreased (A-I)rHDL size and surface charge. The rate of LCAT-mediated cholesterol esterification in (A-I)rHDL varied according to the level of apoA-I glycation and progressively decreased as the extent of apoA-I glycation increased.
  
  
  CONCLUSIONS
  It is concluded that glycation of apoA-I may adversely affect reverse cholesterol transport in subjects with diabetes.
authors.affiliation: Lipid Research Group, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW, 2050, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Nobecourt
authors.forename: M J
authors.initials: MJ
authors.lastname: Davies
authors.forename: B E
authors.initials: BE
authors.lastname: Brown
authors.forename: L K
authors.initials: LK
authors.lastname: Curtiss
authors.forename: D J
authors.initials: DJ
authors.lastname: Bonnet
authors.forename: F
authors.initials: F
authors.lastname: Charlton
authors.forename: A S
authors.initials: AS
authors.lastname: Januszewski
authors.forename: A J
authors.initials: AJ
authors.lastname: Jenkins
authors.forename: P J
authors.initials: PJ
authors.lastname: Barter
authors.forename: K-A
authors.initials: KA
authors.lastname: Rye
mesh_terms: D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry* / Q000502:physiology; D004789:Enzyme Activation; D006031:Glycosylation; D006801:Humans; D006943:Hyperglycemia / Q000097:blood / Q000201:enzymology; D008075:Lipoproteins, HDL / Q000097:blood / Q000302:isolation & purification; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D011765:Pyruvaldehyde / Q000494:pharmacology
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011765:Pyruvaldehyde; D007862:Phosphatidylcholine-Sterol O-Acyltransferase