The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase.
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.
Nobecourt
E
E
Lipid Research Group, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW, 2050, Australia.
Davies
M J
MJ
Brown
B E
BE
Curtiss
L K
LK
Bonnet
D J
DJ
Charlton
F
F
Januszewski
A S
AS
Jenkins
A J
AJ
Barter
P J
PJ
Rye
K-A
KA
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
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011765:Pyruvaldehyde; D007862:Phosphatidylcholine-Sterol O-Acyltransferase