RESUMEN
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.
Asunto(s)
Factores de Crecimiento Endotelial/química , Linfocinas/química , Fragmentos de Péptidos/química , Sitios de Unión , Cristalografía por Rayos X , Cisteína/genética , Factores de Crecimiento Endotelial/genética , Escherichia coli/genética , Linfocinas/genética , Modelos Moleculares , Mutagénesis , Fragmentos de Péptidos/genética , Pliegue de Proteína , Proteínas Tirosina Quinasas Receptoras , Receptores de Factores de Crecimiento , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/química , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module. C75A, C83A, C84A and CC83-84AA variants of t-PA were expressed transiently in human embryonic kidney cells. The biochemical properties of these variants provided experimental evidence to identify the unpaired cysteine in t-PA. Assays of amidolytic activity, plasminogen activation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for comparing these variants with wild-type t-PA. In all assays, C83A t-PA was biochemically equivalent to wild-type t-PA. C75A t-PA, C84A t-PA and CC83-84AA t-PA variants exhibited reduced activities in a variety of functional assays. These variants displayed two-to threefold lower activity in fibrinogen or fibrin stimulated plasminogen activation, and fivefold reduced plasma clot lysis activity compared with that of wild-type t-PA. The affinity of C75A t-PA and C84A t-PA for fibrin was decreased more than two orders of magnitude compared with C83A t-PA or wild-type t-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-fold in mice. The C75A, C84A and CC83-84AA variants displayed significantly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes. These data are consistent with a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as an unpaired sulfhydryl. The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.
Asunto(s)
Cisteína , Variación Genética , Estructura Secundaria de Proteína , Activador de Tejido Plasminógeno/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Sitios de Unión , Línea Celular , Gráficos por Computador , Cricetinae , Embrión de Mamíferos , Factor de Crecimiento Epidérmico/química , Fibrina/metabolismo , Humanos , Riñón , Ratones , Ratones Endogámicos ICR , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Homología de Secuencia de Aminoácido , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/farmacocinética , TransfecciónRESUMEN
Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.
Asunto(s)
Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/metabolismo , Variación Genética , Linfocinas/química , Linfocinas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Cinética , Hígado/metabolismo , Linfocinas/farmacología , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor de Crecimiento Derivado de Plaquetas/química , Mutación Puntual , Estructura Secundaria de Proteína , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Transfección , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Linfocinas/farmacología , Fragmentos de Péptidos/farmacología , Corteza Suprarrenal , Empalme Alternativo , Animales , Unión Competitiva , Células CHO , Capilares , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cricetinae , Endopeptidasas/metabolismo , Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/efectos de los fármacos , Escherichia coli , Fibrinolisina/metabolismo , Humanos , Cinética , Linfocinas/química , Linfocinas/fisiología , Sustancias Macromoleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Polimorfismo Genético , Pliegue de Proteína , Receptores de IgG/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.
Asunto(s)
Inhibidor 1 de Activador Plasminogénico/farmacología , Activador de Tejido Plasminógeno/farmacocinética , Animales , Células CHO , Cricetinae , Fibrina/metabolismo , Tasa de Depuración Metabólica , Mutagénesis Sitio-Dirigida , Inhibidor 1 de Activador Plasminogénico/metabolismo , Estructura Terciaria de Proteína , Conejos , Ratas , Especificidad por Sustrato , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Tyrosylprolylarginyl chloromethyl ketone (YPRck) is a radioiodinatable inhibitor that irreversibly binds the active site of tissue plasminogen activator (tPA). A two-step reaction is employed where (1) the YPRck reagent is iodinated and (2) the 125I-YPRck is reacted with the tPA sample; therefore the oxidative effects of conventional protein iodination are avoided. Using fibrin binding as a probe of native tPA binding function, YPRck labeling was shown to be superior to other types of surface iodination. 125I-YPRck was prepared at a high specific radioactivity; i.e., one 125I per 3.5 molecules of peptidyl chloromethyl ketone. Labeled YPRck formed a one to one covalent, sodium dodecyl sulfate stable, complex with tPA resulting in a preparation of 10 mCi per milligram protein, which corresponded to an incorporation ratio of 1:3.5 (125I-YPRck:tPA). Both one-chain and two-chain forms of tPA reacted with YPRck. Radiolabeling tPA with 125I-YPRck occurred in a time-dependent manner with half-maximal incorporation at approximately 30 min under the conditions employed in these studies. The pH optimum for the reaction of tPA with 125I-YPRck was 7.4. Solutions of tPA at less than 1 microgram/ml were efficiently labeled with 125I-YPRck, thus allowing the quantitation of functional protease by incorporation of radiolabel. Significantly, 125I-YPRck specifically labeled tPA in cell culture supernatants after transient transfection of cells with plasmid DNA containing the gene for tPA. Other serine proteases were tested for their relative reactivity with 125I-YPRck. Thrombin and Factor Xa incorporated 125I-YPRck to higher levels than two-chain tPA; whereas plasmin, urokinase, and other plasma proteases were not as efficiently radiolabeled. The use of 125I-YPRck allows rapid and specific radiolabeling of a large number of tPA samples in a nondenaturing environment with a known localization of the radiolabeling reagent.