,\ô, THE SUBSTRATE SPECIFICITIBS OF HEPATIC LIPASE AND ENDOTHELIAL LIPASE FOR HIGH DENSITY LIPOPROTEIN PHOSPHOLIPIDS: A COMPARATIVE STUDY. My Ngan Duong B.Sc. B.Sc (Hons). A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Department of Medicine Adelaide University Adelaide, Australia. March 2003 11 TABLE OF CONTENTS SUMMARY..... 111 DECLARATION.. .v ACKNOWLEDGEMENTS .vi PUBLICATIONS AND ABSTRACTS vii ABREVIATIONS .ix CHAPTER 1 INTRODUCTION...... 1 CHAPTER 2 MATERIALS AND METHODS........ 52 CHAPTER 3 HYDROLYSIS OF PHOSPHOLIPIDS IN SPMRICAL (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL AND (PDPC)rHDL BY HL AND EL.. 68 CHAPTER 4 HYDROLYSIS OF TRIGLYCERIDES IN SPMRICAL (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL AND (PDPC)rHDL BY HL AND EL... 80 CITPATER 5 TIIE DEVELOPMENT OF A NOVEL SPECTROSCOPIC APPROACH FOR QUANTITATE HL-MEDIATED PHO SPHOLIPID HYDROLYSN 94 CHAPTER 6 CONCLUDING COMMENTS 108 BIBLIOGRAPHY... .111 ll1 S[]MMARY Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family that preferentially hydrolyses high density lipoproteins (HDL) and are involved in the in vivo metabolism and regulation of HDL. Both HL and EL hydrolyses HDL phospholipids. The main HDL phospholipid species are phosphatidylcholines, with the four most abundant being, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine PLPC, 1- palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) and 1-palmitoyl-2- docosahexanoyl phosphatidylcholine (PDPC). The aim of this thesis was to determine if HL and EL have different substrate specificities for HDL phospholipids' In order to carry out these studies, it was important to use HDL that varied systematically in their phospholipid composition, but which were comparable in all other respects. This was achieved by using well-defined, homogenous preparations of spherical reconstituted HDL (rHDL). Although HL and EL both have phospholipase A1 activity, the results showed that HL preferentially hydrolysed rHDL phospholipids with shorter, less saturated sn-2 acyl esters, while EL preferentially acted on rHDL phospholipids with longer, more unsaturated sn-2 acyl esters' To determine if HDL phospholipid composition also affects HL- and El-mediated triglyceride hydrolysis, the rHDL were enriched with radiolabelled triglycerides. As triolein was the only triglyceride constituent in all the rHDL, differences in the rate of triglyceride hydrolysis were attributed to the different phospholipid composition of the rHDL. These studies also gave an indication as to how rHDL phospholipid composition 1V regulated the interactions of HL and EL with the rHDL surface. The results showed that while rHDL phospholipids have a major impact on the interactions of EL with the rHDL surface, this was not the case for the interaction of rHDL and HL. In most of the studies in this thesis formation of nonesterified fatty acids (NEFA) was determined directly using a mass assay. As this approach is not highly sensitive, a novel spectroscopic technique was also developed, whereby NEFA formation was measured as the binding of NEFA to the fluorescent probe AcryloDan-derivatised Intestinal Fatty Acid Binding Protein (ADIFAB). This approach has the advantage of requiring shorter incubation times and smaller amounts of substrates. The results obtained using this approach agreed well with the results that were obtained by measuring NEFA mass. In conclusion, the studies in this thesis show that HL and EL have completely different substrate specificities for rHDL phospholipids. HL preferentially hydrolyses rHDL wirh POPC and PLPC, while EL preferentially hydrolyses rHDL with PAPC and PDPC. The studies also indicate the interactions of EL, but not HL, with the surface of rHDL is regulated by the phospholipid composition of the particles. These findings indicate that HDL phospholipid hydrolysis levels are regulated by the complementary activities of HL and EL. v DECLARATION I certify that this thesis contains no material which has been accepted for the award of any other degree of diploma in any university or any other tertiary institutions and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying. MvN Duong. VI ACKNOWLEGEMENTS I wish to thank my principal supervisor, Associate-Professor Kerry-Anne Rye for her expert supervision throughout this work, which was performed at the Lipid Research Laboratory, Hanson Institute in Adelaide. Her generous support and encouragement have been invaluable to this work. I would also like to thank my co-supervisor, Professor Philip Barter, for his enthusiastic council and insightful guidance My gratitude to my colleges for their friendship and advice; Dr. Moira Clay, Dr. Paul Baker, Dr. Neil Hime, Richard Bright, Jane Roberts, Maria Psaltis, Kate Drew, Dr. Stephen Nicholls, Dr. Patrick Kee, Dr. Daniela Caiazza, Diana Pyle, Dr. Dale Ashby, Dr. Anisa Jahangiri, Kevin Wee, Angel Liang, Nongnuch Settasatian and Mary Marucci I thank the Helpman Scholarship in Cardiovascular Research for supporting me during my studies Finally, my heartfelt thanks to my parents and family for their patience and support that have never wavered throughout this work. vll PUBLICATIONS AND ABSTRACTS Publications Duong M, Psaltis M, Rader D, Marchadier D, Barter PJ, and Rye KA. (2003) Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high density lipoproteins. In the process of being submitted. Rye KA, Duong M, Psaltis M, Curtiss L, Bonnet D, Stocker R and Barter PJ' (2002) Evidence that phospholipids play a key role in pre-p apoA-I formation and high density lipoprotein remodeling . Biochemistry 4l:12538-12545. Other Publications Settasatian N, Duong M, Curtiss LK, Ehnholm C, Jauhiainen M, Huuskonen J and Rye KA. (2001) The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein. Journal of Biotogical Chemistry 276(29): 26898-905. Rye KA and Duong M. (2000) Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins. Journal of Lipid Research 41(10): 1640-50' Abstracts Presented MN.Duong, PJ. Barter and K-A. Rye. 1997. The influence of phospholipids on the size and structure of spherical, reconstituted high density lipoproteins. XIth International Symposium of Atherosclerosis, Paris, France. v1ll MN.Duong, W. van Amelsfort, H-Q. Liang and K-4. Rye. 1998. The influence of phospholipid concentration on the size, structure and function of reconstituted high density lipoproteins (HDL). XXIVth Annual Scientific Meeting of the Australian Athe ro s clero si s S o ciety, C aints, Que ensland, Austr alia. MN.Duong and K-4. Rye. 2000. The influence of phospholipid acyl chain composition on hepatic lipase-mediated hydrolysis of high density lipoprotein phospholipid. XIIth Int e rnati onal Symp o s ium of Athe r o s cl e r o s i s, S t o ckholm, Sw e den. MN. Duong, D. Rader, PJ. Barter and K-4. Rye. 2001. Evidence that endothelial lipase and hepatic lipase have opposing substrate specificities. XXVIth Annual Scientific Meeting of the Australian Atherosclerosis Society, Fremantle, Western Australia. IX ABBRBVIATIONS cr-helices amphipathic helices ABCAI ATP binding cassette transporter apo apolipoprotein BITT butylated hydroxytoluene BSA bovine serum albumin CAD coronary artery diseases CE cholesteryl esters CETP cholesteryl ester transfer protein CHD coronary heart disease d density DETAPAC Diethylenetriamine pentaacetic acid DTNB 5, 5' -Dithio-bis(nitrobenzoic acid) EDTA-Naz ethylenediaminetetraacetic acid, disodium salt EL endothelial lipase FED fish-eye disease FH familial hypercholesterolemi a FLD familial LCAT deficiency FPLC fast performance liquid chromatograpy GPLRP2 guinea pig pancreatic lipase-related protein 2 HDL high density lipoproteins HL hepatic lipase hPL human pancreatic lipase IDL intermediate density lipoproteins X LCAT lecithin : cholesterol acyltransferase LDL low density lipoproteins LPL lipoprotein lipase lyso-PC lysophosphatidylcholines NEFA nonesterified fatty acid NF-KB nuclear factor rB PAPC 1 -p almi toy l-2 - ar achidonyl ph o sph ati dyl c holine PDPC 1 -palmitoyl-2-docosahexanoyl phosphatidylcholine PLAz phospholipase Az PLPC 1 -palmitoyl-2-linoleoyl phosphatidylcholine PLTP phospholipid transfer protein PON paraoxonase POPC 1 -palmitoyl-2-oleoyl phosphatidylcholine RCT reverse cholesterol transPort rHDL reconstituted HDL SAA serum amyloid A SDS sodium dodecyl sulphate SR-BI scavenger receptor class B tYPe I TBS Tris-buffered saline TC total cholesterol TG triglycerides TO triolein UC unesterified cholesterol VLDL very low density lipoproteins CHAPTER 1. INTRODUCTION LIPOPROTEINS 1.1 PLASMA LIPID TRANSPORT t.2 CLASSIFICATION OF LIPOPROTEINS 1.3 STRUCTURE AND COMPOSITION OF LIPOPROTEINS Surface Constituents PhospholiPids ApoliPoProteins Core Constituents CholesterYl Esters Triglycerides r.4 LIPOPROTEIN METAB OLISM 1.5 LIPOPROTEINS AND ATI{EROSCLEROSIS Proatherogenic LiPoProteins Antiatherogenic LiPoProteins r.6 ANTIATI{EROGENIC PROPERTIES OF HDL Reverse Cholesterol TransPort Antioxidant ProPerties of HDL Anti-infl ammatory Properties of HDL t.7 HDL PHOSPHOLIPIDS 1.8 PLASMA FACTORS THAT REMODEL HDL 1.8.1 Lecithin:cholesterol Acyltransferase (LCAT) r.8.2