Hepatic lipase and dolichol esterification

Abstract: Doctoral Dissertation 1997 Clinical Research Center, Novum and Department of Neuroscience Karolinska Institutet Stockholm, Sweden Dolichol is one of the mevalonate pathway end products, present in all tissuesand cellular membranes. The lipid is a primary alcohol consisting of 16-23 isoprenoidresidues and occurs as a free alcohol, monophosphate or fatty acyl ester. The phosphorylatedform functions as an obligatory intermediate in the biosynthesis of N-linked oligosaccharidesof glycoproteins. Studies in model membranes indicate that dolichol has a destabilizingeffect on phospholipid bilayers by increasing membrane fluidity and permeability.The aim of the present investigation was to identify and characterize the enzymesinvolved in dolichol esterification and thereby gain insight into the possible functionof this lipid. When phosphatidylcholine:phosphatidylethanolamine liposomes containing 2% dolicholwere injected into rats, the polyisoprenoid lipid was esterified in the circulation.Preheparinization of the animal greatly increased the esterification and it was concludedthat an enzyme was liberated into the plasma by heparin. It was further demonstratedthat LCAT was not responsible for this activity and that human postheparin plasmaalso contained a dolichol acylating activity. The enzyme was partially purified from heparinized liver perfusates and characterizedwith respect to its substrate specificity. It was shown that the enzyme was a transacylaseand that phosphatidyl-ethanolamine and -serine were by far the preferred acyl donors.Only fatty acids in sn-1 position were utilized and cholesterol and retinol werenot esterified. The use of Brij-35 instead of Triton X-100 for delipidization of the enzyme madeit possible to purify the enzyme to homogeneity without loosing the dolichol transacylaseactivity. The protein had a molecular mass of 61 kDa and was shown to be identicalwith hepatic lipase. The hepatic lipase-catalyzed dolichol esterification was stronglystimulated by a plasma cofactor that also shifted the pH optimum from 8.5 to 7.5.Gel filtration of whole plasma indicated that the cofactor might be associated withHDL. The cofactor was purified from the lipoprotein-free high density fraction of plasmaand the identity with apolipoprotein (apo) A-IV was demonstrated. The role of apoA-IV in hepatic lipase-catalyzed hydrolysis of human HDL-2 and VLDL was investigated.The cofactor strongly stimulated phospholipid hydrolysis and completely inhibitedtriglyceride hydrolysis in both lipoproteins. Thus, apo A-IV is an important cofactorto hepatic lipase, and it is suggested that apo A-IV rich HDL is a preferred lipoproteinsubstrate for hepatic lipase. 1997 Pavel J. Sindelar ISBN 91-628-2772-3 AkademitryckAB, Edsbruk 1997