RESUMEN
Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.
Asunto(s)
Autoantígenos/metabolismo , Colágeno Tipo IV , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Fragmentos de Péptidos , Receptores de Vitronectina/metabolismo , Alquilación , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/química , Autoantígenos/farmacología , Caspasa 3 , Caspasas/metabolismo , Bovinos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/química , Colágeno/farmacología , Disulfuros/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Proteínas de la Matriz Extracelular/química , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , Vitronectina/metabolismoRESUMEN
The existence of two homologous mannose 6-phosphate receptors (MPRs) with overlapping, but distinct functions has raised the question of at what stage in the phylogenetic tree the two receptors have occurred for the first time. In this paper, we present a partial cDNA sequence of Mr 300 kDa MPR (MPR 300) from poeciliid fish (Xiphophorus). It contains a 5'-untranslated region followed by the initiator ATG, and an open reading frame that corresponds to cassettes 1-5 and part of cassette 6 of mammalian MPR 300. The size of the mRNA transcript for fish MPR 300 was comparable with that of other vertebrates. The amino acid sequence of fish MPR 300 displays 48-52% similarity with mammalian and chicken MPR 300. In particular, all the cysteine residues involved in disulfide bonding and an arginine residue, which is considered to be part of the mannose 6-phosphate binding site in cassette 3 of mammalian MPR 300, are conserved. Sequence similarities were significantly higher within cassette 3 and within cassette 5, to which a ligand-binding function has not yet been ascribed. Sequence similarities of the internal cassettes of MPR 300 are discussed with regard to the multifunctional nature of MPR 300.
Asunto(s)
Secuencia Conservada/genética , ADN Complementario/análisis , Poecilia/genética , Receptor IGF Tipo 2/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Bovinos , Pollos , Cartilla de ADN/química , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Mannose 6-phosphate receptors (MPRs) are known to occur in mammals, birds, reptiles and amphibians. Here we provide evidence for the presence of two MPRs in fish, the earliest vertebrates. Using phosphomannan-Sepharose affinity chromatography, MPR 300 was purified from liver membrane extract of trout. The purified trout liver MPR 300 showed similar electrophoretic mobility as the goat liver receptor and a pH optimum of 7.0 for binding to phosphomannan. The presence of MPR 46 in fish was shown by metabolically labelling embryonic fish cells (Xiphophorus) and immunoprecipitation with an antibody against the cytoplasmic tail of human MPR 46 (anti-MSC1). This antibody had recently been shown to immunoprecipitate MPR 46 also from reptiles and amphibians.
Asunto(s)
Receptor IGF Tipo 2/química , Animales , Evolución Molecular , Peces , Concentración de Iones de Hidrógeno , Hígado/metabolismo , Mananos/metabolismo , Proteínas de la Membrana/química , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas/químicaRESUMEN
Mannose 6-phosphate receptor (MPR) proteins designated as MPR 300 and MPR 46 have earlier been purified from some mammals on phosphomannan coupled to cyanogen bromide activated Sepharose. In a recent study, the goat liver MPR 300 has been directly purified using Sepharose-divinylsulfone-pentamannosyl phosphate matrix (Sivakumar N. 1996, J. Biochem. Biophys. methods, 31, 181-184(1)). In the present report, we describe the preparation of another affinity matrix Sepharose-divinylsulfone-phosphomannan and its utility in purifying the MPR proteins from goat liver. While the MPR 300 from goat liver showed an electrophoretic mobility similar to other mammalian MPRs, the small receptor showed a molecular weight of 36 kDa. Antibodies raised against goat liver MPR 300 react specifically with the large receptor protein. Additionally affinity purified peptide specific antibody corresponding to amino-acid residues 26-42 (ADGCDFVCRSKPRNVPA) of the cytoplasmic tail of the human liver MPR 46 (Pohlmann et al, 1988. Proc. Natl. Acad. Sci. USA, 84, 5575-5579 (2)) cross-reacts with the purified small receptor.