title: High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein.
abstract: Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation.
authors.affiliation: Institute of Medical Biochemistry, Karl-Franzens University, Graz, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Frank
authors.forename: K
authors.initials: K
authors.lastname: Krasznai
authors.forename: S
authors.initials: S
authors.lastname: Durovic
authors.forename: E M
authors.initials: EM
authors.lastname: Lobentanz
authors.forename: H
authors.initials: H
authors.lastname: Dieplinger
authors.forename: E
authors.initials: E
authors.lastname: Wagner
authors.forename: K
authors.initials: K
authors.lastname: Zatloukal
authors.forename: M
authors.initials: M
authors.lastname: Cotten
authors.forename: G
authors.initials: G
authors.lastname: Utermann
authors.forename: G M
authors.initials: GM
authors.lastname: Kostner
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002461:Cell Line, Transformed; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D018082:Kringles / Q000235:genetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D014162:Transfection
chemical_list: D001053:Apolipoproteins; D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; D001120:Arginine; D003545:Cysteine