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.

No abstract

No abstract

Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug`s target, topoisomerase IIα (topo IIα). It is not known, however, whether a cytoplasmic pool of topo IIα is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo IIα fused to GFP and have examined the transformants for the subcellular localization of topo IIα and their resistance to etoposide. Transient transfection resulted in high‐level expression of all GFP‐topo IIα fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy‐terminal topo IIα domain (aa 428‐1504, 639‐1028 or 1028‐1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP‐topo IIα proteins were observed in cell lines expressing constructs that retain the amino‐terminus of the enzyme (aa 1‐1214, aa 1‐939, aa 1‐611). Our results suggest that the topo IIα amino‐terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo IIα also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo IIα is not sufficient to render cultured mammalian cells drug resistant. Int. J. Cancer 88:99–107, 2000. © 2000 Wiley‐Liss, Inc.

No abstract