Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains.
Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.
Klose
R
R
Institute for Medical Biology and Human Genetics, Universität Innsbruck, 6020 Innsbruck, Austria.
Fresser
F
F
Kochl
S
S
Parson
W
W
Kapetanopoulos
A
A
Fruchart-Najib
J
J
Baier
G
G
Utermann
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053241:Apoprotein(a); D001665:Binding Sites; D016466:CHO Cells; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D017270:Lipoprotein(a) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014407:Tumor Cells, Cultured
D001053:Apolipoproteins; D017931:DNA Primers; D017270:Lipoprotein(a); D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005337:Fibrin; D005340:Fibrinogen; D053241:Apoprotein(a)