title: Secretion of active human lecithin-cholesterol acyltransferase by insect cells infected with a recombinant baculovirus.
abstract: Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.
authors.affiliation: University Department of Medicine, Royal Free Hospital School of Medicine, London, U.K.
authors.forename: D
authors.initials: D
authors.lastname: Chawla
authors.forename: J S
authors.initials: JS
authors.lastname: Owen
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests
chemical_list: D018076:DNA, Complementary; D007862:Phosphatidylcholine-Sterol O-Acyltransferase