RESUMO
Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator.
Assuntos
Angiopoietinas/metabolismo , Lipase Lipoproteica/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Angiopoietinas/isolamento & purificação , Animais , Biocatálise , Bovinos , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas , Ativação Enzimática , Heparina , Temperatura Alta , Lipase Lipoproteica/isolamento & purificação , Lipase Lipoproteica/metabolismo , Microscopia de Força Atômica , Modelos Biológicos , Sefarose , Fatores de TempoRESUMO
Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.
Assuntos
Angiopoietinas/genética , Angiopoietinas/imunologia , Angiopoietinas/isolamento & purificação , Anticorpos/isolamento & purificação , Engenharia de Proteínas/métodos , Proteína 1 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Glutationa Transferase/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The Tie1 and Tie2 receptor tyrosine kinases and the Tie2 ligands, the angiopoietins, play critical roles in vertebrate vascular embryogenesis, helping to mediate the interaction between endothelial cells and the pericytes or vascular smooth muscle cells that envelop and support them. We have obtained full-length cDNA sequences for zebrafish orthologs of angiopoietin-1 (ang1), angiopoietin-2 (ang2), and angiopoietin-like-3 (angptl3). Ang1 is expressed in head ventral mesenchyme, in the ventromedial region of somites, in mesenchyme surrounding trunk axial vessels, and in the hypochord, a transient embryonic structure of endodermal origin that has been implicated in dorsal aorta assembly in both zebrafish and Xenopus. Ang2 is expressed in head and anterior trunk ventral mesenchyme and the developing pronephric glomeruli. Angptl3 is expressed in the yolk syncytial layer.