A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping

Abstract

Apolipoprotein[a] phenotyping is a critically important method to explore the role of kringle-4 repeat number as a modulator of lipoprotein[a]-associated cardiovascular risk. The availability of a kringle-4 number-based reference standard is therefore necessary for a reliable and generally accepted classification of apo[a] phenotypes. We propose here a battery of recombinant apo[a] isoforms that may be used as the reference standard in various gel systems. Five plasmids encoding for r-apo[a] containing a known number (n ‫؍‬ 9, 13, 17, 25, 33) of plasminogen-like kringle-4 copies were constructed, and transfected into the human embryonic kidney cell line 293. The electrophoretic mobility of the recombinant apo[a] isoforms expressed by these cells in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotting using an antibody specific for human apo[a]. The equation of the linear relationship between log r-apo[a] kringle number and relative migration was used to determine the isoform size of apo[a] in normal human plasma. A very good correlation ( r ‫؍‬ 0.97) was found with the genotype (pulsed-field gel eletrophoresis of kpn I-digested restriction fragments of genomic DNA) and among electrophoretic methods. The proposed recombinant standard offers the possibility to identify apo[a] isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic techniques and a nomenclature based on its molecular structure, i.e., the number of kringle-4 repeats.-Anglés-Cano, E., S. Loyau, G. Cardoso-Saldaña, R. Couderc, and P. Gillery. A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping.

Interaction of Fibrin(ogen) with Apolipoprotein(a): Further Characterization and Identification of a Novel Lysine-Dependent Apolipoprotein(a)-Binding Site within the γ Chain 287−411 Region

“…The recombinant γ module, including human fibrinogen γ chain residues 148-411 and its subfragments, γ148-191, γ192-286, and γ287-411, were prepared as described in refs 18 and 19. Recombinant apo(a) A10 was expressed and purified as described previously (20). Fibrinogen-derived fragments D 1 and E 3 were prepared by the procedures described in ref 21.…”

Evidence for several independent genetic variants affecting lipoprotein (a) cholesterol levels

“…Protein concentrations were measured with the Pierce BCA kit (Thermo Scientific, Rockford, IL). Of note, 66 µg of liver protein was subjected to electrophoresis on NuPage 3-8% Tris-Acetate Gel (Life Technologies, Grand Island, NY), samples were transferred to nitrocellulose membrane, and then immunoblotted with Lp(a) sheep polyclonal antibody ( provided by Dr Eduardo Angles-Cano, Institut national de la santé et de la recherche médicale, France) at a dilution of 1:20 000 for 2 h at room temperature (51). Then the blots were incubated with a donkey anti-sheep IgG secondary antibody conjugated with peroxidase (Santa Cruz Biotechnology, Dallas, Texas) at a dilution of 1:20 000 for 1 h at room temperature.…”

Assessment of Apolipoprotein(a) Isoform Size Using Phenotypic and Genotypic Methods

“…Variations in the number of LPA KIV2 repeats can be estimated via immunoblotting electrophoresis or the pulsed-field electrophoresis of unamplified genomic DNA. Immunoblotting electrophoresis is used to detect apo(a) protein isoforms [ 40 ], whereas the pulsed-field electrophoresis of unamplified genomic DNA is used to detect variations in the LPA gene [ 41 ]. These experimental methods are both technically challenging, laborious, and time-consuming.…”