RESUMEN
Urokinase plasminogen activator (uPA) and its receptor are key components of a cell surface proteolytic cascade used by tumor cells and capillary endothelial cells for basement membrane invasion, a process required for metastasis and angiogenesis. We have cloned, expressed, and purified the epidermal growth factor-like domain of murine uPA alone and fused it to the Fc portion of human IgG as high-affinity murine urokinase receptor antagonists. These molecules are potent inhibitors of murine urokinase binding to its receptor and inhibit angiogenesis in an in vitro model of capillary tube formation in fibrin gels. In vivo, basic fibroblast growth factor-induced neovascularization and B16 melanoma growth in syngeneic mice are also substantially suppressed by these molecules. Coupled with previous studies showing inhibition of metastasis, these findings suggest that urokinase receptor antagonists may be useful therapeutically as inhibitors of tumor progression.
Asunto(s)
Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Receptores de Superficie Celular/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Colágeno , ADN Complementario/genética , Combinación de Medicamentos , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Epítopos/química , Epítopos/genética , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genes de Inmunoglobulinas , Humanos , Inmunoglobulina G/genética , Laminina , Linfocinas/farmacología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteoglicanos , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The human urokinase plasminogen activator (uPA) epidermal growth factor-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast. Purified human uPA 1-48 and uPA 1-48glu complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.
Asunto(s)
Bacteriófagos/genética , Factor de Crecimiento Epidérmico/química , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/genética , Activador de Plasminógeno de Tipo Uroquinasa/química , Secuencia de Aminoácidos , Bacteriófagos/metabolismo , Secuencia de Bases , Unión Competitiva , Cartilla de ADN , Factor de Crecimiento Epidérmico/genética , Epítopos/inmunología , Genes Sintéticos , Humanos , Ligandos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/aislamiento & purificación , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy expression vector in Escherichia coli under control of the tac promoter. The resulting plasmid construction produced 3C proteinase as a soluble and active enzyme constituting approximately 10% of total cellular proteins. The enzyme was purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was developed, and synthetic peptides derived from the predicted cleavage sites of the HAV polyprotein were tested for proteolysis of the enzyme. The peptide representing the 2B/2C cleavage site was cleaved most efficiently with a Km and kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed mutagenesis was then used to identify the cysteine at position 172 as the active site nucleophile. Finally, the purified enzyme showed the expected endoproteinase activity on the P1 precursor protein generated by in vitro transcription/translation.
