Lipophorin structure analyzed by in vitro treatment with lipases.
Adult Manduca sexta high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I, -II, and -III) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-III. The amount of phospholipid remains constant in lipophorin (140 mol/mol of lipophorin), while the diacylglycerol content varies between different lipophorin species (310 mol/mol HDLp up to 1160 mol/mol LDLp). Both lipophorin particles were enzymatically depleted of phospholipid or diacylglycerol by in vitro incubation with either phospholipase A2 or triacylglycerol lipase. Albumin was used to remove free fatty acids generated during the reaction. Treatment with phospholipase A2 removed all phospholipids (except sphingomyelin) and the resulting particles were stable. Triacylglycerol lipase hydrolyzed large fractions of diacylglycerol. The resulting particles were smaller in size, higher in density, and devoid of apoLp-III. The particles retained apoLp-I and -II and the other lipid components, including a substantial amount of diacylglycerol. Structural integrity of diacylglycerol-depleted lipophorin was confirmed by electron microscopical analysis. When treated with both phospholipase A2 and triacylglycerol lipase, lipophorin precipitated. From these results we conclude that: 1) all phospholipid and apoLp-III are located at the surface of lipophorin, whereas diacylglycerol is partitioned between the sublayers and the surface of the particle; 2) both diacylglycerol and phospholipid play a role in stabilizing lipophorin in the aqueous medium; and 3) lipophorin can be extensively unloaded and still retain its basic structure, a necessary feature for its function as a reusable lipid shuttle.
Kawooya
J K
JK
Department of Biochemistry, University of Arizona, Tucson 85721.
van der Horst
D J
DJ
van Heusden
M C
MC
Brigot
B L
BL
van Antwerpen
R
R
Law
J H
JH
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008074:Lipoproteins; D008854:Microscopy, Electron; D009036:Moths; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2
D002352:Carrier Proteins; D004075:Diglycerides; D008055:Lipids; D008074:Lipoproteins; C032830:lipophorin; D008049:Lipase; D010741:Phospholipases A; D054467:Phospholipases A2