RESUMO
Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.
Assuntos
Apolipoproteínas A/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Apolipoproteínas A/química , Apolipoproteínas A/genética , Arteriosclerose/metabolismo , Sequência de Bases , Sítios de Ligação , Células COS , Artérias Carótidas/metabolismo , DNA Complementar/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Kringles , Lipoproteína(a)/química , Lipoproteína(a)/genética , Lipoproteína(a)/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Apolipoproteínas/química , Apolipoproteínas/metabolismo , Fibrina/química , Fibrina/metabolismo , Lipoproteína(a)/química , Lipoproteína(a)/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteínas/genética , Apoproteína(a) , Sítios de Ligação , Células CHO , Clonagem Molecular , Sequência Conservada , Cricetinae , Primers do DNA , Fibrinogênio/química , Fibrinogênio/metabolismo , Biblioteca Gênica , Genes Reporter , Humanos , Lipoproteína(a)/genética , Fígado/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Células Tumorais CultivadasRESUMO
Using differential screening of cytokine-activated versus resting porcine aortic endothelial cells (PAEC), we have isolated a member of the family of Ras/GTP-binding proteins. The cDNA encodes a 34-kilodalton protein showing 97% homology to Gem, a gene recently isolated from activated T cells, likely representing its porcine homologue. The amino acid sequence differs from the Ras consensus by the absence of a C-terminal isoprenylation site and a glycine to glutamic acid substitution in the third GTP-binding domain. We report here, that pigGem mRNA is strongly inducible in PAEC upon activation by either IL-1 alpha, TNF alpha or lipopolysaccharide (LPS). Low constitutive expression is found in several organs. Epitope-tagged pigGem transfected into endothelial cells (EC) localizes to the cytoplasm and to the inner side of the plasma membrane. Structural features of Gem and its inducibility apparently restricted to T cells and endothelial cells, together with Rad, a GTPase overexpressed in skeletal muscle cells of type II diabetic individuals, define a new branch within the superfamily of GTP-binding proteins.
