title: Identification of two functionally distinct lysine-binding sites in kringle 37 and in kringles 32-36 of human apolipoprotein(a).
abstract: The well documented association between high plasma levels of lipoprotein(a) (Lp(a)) and cardiovascular disease might be mediated by the lysine binding of apolipoprotein(a) (apo(a)), the plasminogen-like, multikringle glycoprotein in Lp(a). We employed a mutational analysis to localize the lysine-binding domains within human apo(a). Recombinant apo(a) (r-apo(a)) with 17 plasminogen kringle IV-like domains, one plasminogen kringle V-like domain, and a protease domain or mutants thereof were expressed in the human hepatocarcinoma cell line HepG2. The lysine binding of plasma Lp(a) and r-apo(a) in the culture supernatants of transfected HepG2 cells was analyzed by lysine-Sepharose affinity chromatography. Wild type recombinant Lp(a) (r-Lp(a)) revealed lysine binding in the range observed for human plasma Lp(a). A single accessible lysine binding site in Lp(a) is indicated by a complete loss of lysine binding observed for r-Lp(a) species that contain either a truncated r-apo(a) lacking kringle IV-37, kringle V, and the protease or a point-mutated r-apo(a) with a Trp-4174-->Arg substitution in the putative lysine-binding pocket of kringle IV-37. Evidence is also presented for additional lysine-binding sites within kringles 32-36 of apo(a) that are masked in Lp(a) as indicated by an increased lysine binding for the point mutant (Cys-4057-->Ser), which is unable to assemble into particles. An important role of these lysine-binding site(s) for Lp(a) assembly is suggested by a decreased assembly efficiency for deletion mutants lacking either kringle 32 or kringles 32-35.
authors.affiliation: Department of Molecular Biology, Boehringer Mannheim GmbH, Federal Republic of Germany.
authors.forename: A
authors.initials: A
authors.lastname: Ernst
authors.forename: M
authors.initials: M
authors.lastname: Helmhold
authors.forename: C
authors.initials: C
authors.lastname: Brunner
authors.forename: A
authors.initials: A
authors.lastname: Pethö-Schramm
authors.forename: V W
authors.initials: VW
authors.lastname: Armstrong
authors.forename: H J
authors.initials: HJ
authors.lastname: Müller
mesh_terms: D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D053241:Apoprotein(a); D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002846:Chromatography, Affinity; D003545:Cysteine; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D018082:Kringles; D017270:Lipoprotein(a); D008113:Liver Neoplasms; D008239:Lysine; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014162:Transfection; D014364:Tryptophan; D014407:Tumor Cells, Cultured
chemical_list: D001053:Apolipoproteins; D018076:DNA, Complementary; D017270:Lipoprotein(a); D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D014364:Tryptophan; D053241:Apoprotein(a); D008239:Lysine; D003545:Cysteine