RESUMEN
BACKGROUND: Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. METHODS AND RESULTS: Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE-/- mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. CONCLUSIONS: These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.
Asunto(s)
Apolipoproteínas B/inmunología , Arteriosclerosis/prevención & control , Inmunización Pasiva , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Región Variable de Inmunoglobulina/uso terapéutico , Lipoproteínas LDL/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteína B-100 , Apolipoproteínas B/química , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arteriosclerosis/patología , Evaluación Preclínica de Medicamentos , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/genética , Lipoproteínas LDL/farmacología , Macrófagos/patología , Masculino , Malondialdehído/química , Malondialdehído/inmunología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Monocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa(3), which is a quinol oxidase, and cytochrome caa(3), which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, Cu(A). B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa(3) but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa(3). B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the Cu(A) centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.