Cloning and expression analysis of a cDNA encoding lipoprotein lipase from the liver of adult grass carp (Ctenopharyngodon idella)

Abstract

AbstractA full-length cDNA coding lipoprotein lipase (LPL)was cloned from the liver of adult grass carp(Ctenopharyngodon idella) using reverse transcriptionpolymerasechainreaction(RT-PCR)andrapidampli-¢cationofcDNAendsapproaches.ThecDNAobtainedwas 2414bp long with a1524bp open reading frameencoding507aminoacids,includingaputativesignalpeptide 21 amino acids long. The LPL protein has acalculated molecular weight of 57.77kDaandan iso-lectric point of 8.132.The maindomains of LPL, suchas catalytic site, disulphide bridge, N-linked glycosy-lationsite,heparin-bindingdomain,lipid-bindingsiteand site of dimer formation, are basically conservedbetweenthegrasscarpandothervertebrates.Thetis-sue distribution of LPL mRNA in the liver, head kid-ney, mesenteric adipose tissue, heart and whitemuscle of adult grass carp was analysed using thesemi-quantitative RT-PCR methodusing b-actingeneas an internal control; the result showed that the ex-pressionsofLPLmRNAweredetectedinallexaminedtissues of adult grass carp. The expression levels ofLPLinthemesentericadiposetissuewerethehighestamong these tissues, followed by the liver and headkidney and the lowest expression was found in theheartandwhitemuscle.Keywords: Ctenopharyngodon idella, full-lengthcDNA,lipoproteinlipasegene, RACEIntroductionLipoprotein lipase (LPL, EC 3.1.1.34) is a member ofthelipasegenesuperfamily, whichinitiallyconsistedof three mammalian lipases [pancreatic lipase (PL);lipoprotein lipase; and hepatic lipase (HL)] based onamino acid sequence similarity and gene organiza-tion (Saera-Vila, Calduch-Giner, Gomez-Requeni,Medale, Kaushik & Perez-Sanchez 2005). Family sizeincreased when several proteins were subsequentlyadded based on amino acid homology, includingPL-relatedproteins1and2,phosphatidylserinephos-pholiaseA1and endothelial lipase (EL). All these en-zymes would have evolved from a common ancestorand they play a critical role in the absorption andmetabolism of lipids and lipoproteins. Lipases arewater-soluble enzymes that hydrolyse ester bonds ofwater-insoluble substrates such as triglycerides,phospholipids andcholesteryl esters (Wong & Schotz2002; Mukherjee2003).Mammalian mature LPL is a glycoprotein that isprimarily synthesized in adipocytes, muscle cells,macrophages and lactating mammary gland, butnot in the adult liver (Wion, Kirchgessner, Lusis,Schotz & Lawn 1987; Raisonnier, Etienne, Arnault,Brault, Noe, Chuat & Galibert1995).The human LPLgeneencoded475aminoacids,includinga27aminoacid signal peptide.The mature LPL protein consistsof 448 amino acids with a molecular weight of55kDa after resecting the signal peptide (Wong &Schotz2002).Theenzymeisactiveasanon-covalenthomodimerandisboundtothesurfaceofendothelialcells via glycosaminoglycans, from where it can bereleased byheparin (Mead, Irvine & Ramji 2002). Li-poprotein lipase plays a key role in lipid metabolismby hydrolysing triglycerides from chylomicrons andvery low-density lipoproteins (VLDL) to non-esteri-¢ed fatty acids and 2-monoacylglycerol, which areutilized either for storage or for energy production(Wang, Hartsuck& McConathy1992).The abnormal-