RESUMEN

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V(H) domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V(H) domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.

Asunto(s)

Disulfuros/química , Inmunoglobulina G/química , Región Variable de Inmunoglobulina/química , Anticuerpos Antiidiotipos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Cromatografía por Intercambio Iónico , Disulfuros/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Omalizumab , Papaína/metabolismo , Procesamiento Proteico-Postraduccional , Ácido Pirrolidona Carboxílico/química , Proteínas Recombinantes/química

RESUMEN

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/química , Factores de Crecimiento Nervioso/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Receptor de Factor de Crecimiento Nervioso/química

RESUMEN

Angiogenesis is required for tumor growth and metastasis, and inhibition of angiogenesis is a promising approach for anticancer therapy. Tie2 (a.k.a Tek) is an endothelium-specific receptor tyrosine kinase known to play a role in tumor angiogenesis. To explore the therapeutic potential of blocking the Tie2 pathway, an adenoviral vector was constructed to deliver a recombinant, soluble Tie2 receptor (AdExTek) capable of blocking Tie2 activation. Two days after i.v. injection of AdExTek, the plasma concentration of ExTek exceeded 1 mg/ml and was maintained for about 8 days. Administration of AdExTek to mice with two different well established primary tumors, a murine mammary carcinoma (4T1) or a murine melanoma (B16F10.9), significantly inhibited the growth rate of both tumors (64% and 47%, respectively). To study the effect of ExTek on tumor metastasis, both tumor cell lines were coinjected i.v. with either AdExTek or a control virus. Mice coinjected with control virus developed numerous large, well vascularized lung metastases. In contrast, mice coinjected with AdExTek virus developed few, if any, grossly apparent metastases, and histologic examination revealed only small avascular clusters of tumor cells. Administration of AdExTek also inhibited tumor metastasis when delivered at the time of surgical excision of primary tumors in a clinically relevant model of tumor metastasis. This study demonstrates the potential utility of gene therapy for systemic delivery of an antiangiogenic agent targeting an endothelium-specific receptor, Tie2.

Asunto(s)

Endotelio Vascular/enzimología , Terapia Genética , Neovascularización Patológica/genética , Proteínas Tirosina Quinasas Receptoras/genética , Adenoviridae/genética , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Femenino , Humanos , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Melanoma Experimental/terapia , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/genética , Fosforilación , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Células Tumorales Cultivadas

RESUMEN

Angiogenesis is thought to depend on a precise balance of positive and negative regulation. Angiopoietin-1 (Ang1) is an angiogenic factor that signals through the endothelial cell-specific Tie2 receptor tyrosine kinase. Like vascular endothelial growth factor, Ang1 is essential for normal vascular development in the mouse. An Ang1 relative, termed angiopoietin-2 (Ang2), was identified by homology screening and shown to be a naturally occurring antagonist for Ang1 and Tie2. Transgenic overexpression of Ang2 disrupts blood vessel formation in the mouse embryo. In adult mice and humans, Ang2 is expressed only at sites of vascular remodeling. Natural antagonists for vertebrate receptor tyrosine kinases are atypical; thus, the discovery of a negative regulator acting on Tie2 emphasizes the need for exquisite regulation of this angiogenic receptor system.

Asunto(s)

Vasos Sanguíneos/metabolismo , Endotelio Vascular/citología , Neovascularización Fisiológica , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Secuencia de Aminoácidos , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Vasos Sanguíneos/embriología , Células Cultivadas , Clonación Molecular , Embrión de Mamíferos/metabolismo , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/metabolismo , Femenino , Humanos , Ligandos , Linfocinas/genética , Linfocinas/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Proteínas/química , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Mammalian cells constantly monitor and respond to a myriad of extracellular signals, often by using cell surface receptors. Two important classes of cell surface receptors include the receptor tyrosine kinases, which recognize peptide growth factors such as insulin, and the integrins, which most often mediate binding to components of the extracellular matrix. We report that the collagens serve as ligands for the previously orphan family of discoidin domain-containing receptor-like tyrosine kinases. The unexpected realization that an extracellular matrix molecule can directly serve as a ligand for receptor tyrosine kinases provides an example of ligands shared by integrins and receptor tyrosine kinases, and this finding seems likely to change prevailing views about the mechanisms by which cells perceive and respond to the extracellular matrix.

Asunto(s)

Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Células COS , Colágeno/farmacología , Receptores con Dominio Discoidina , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Integrinas/metabolismo , Ligandos , Fosforilación , Plásmidos , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Mitogénicos/química , Receptores Mitogénicos/genética , Rabdomiosarcoma , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

TIE2 is a receptor-like tyrosine kinase expressed almost exclusively in endothelial cells and early hemopoietic cells and required for the normal development of vascular structures during embryogenesis. We report the identification of a secreted ligand for TIE2, termed Angiopoietin-1, using a novel expression cloning technique that involves intracellular trapping and detection of the ligand in COS cells. The structure of Angiopoietin-1 differs from that of known angiogenic factors or other ligands for receptor tyrosine kinases. Although Angiopoietin-1 binds and induces the tyrosine phosphorylation of TIE2, it does not directly promote the growth of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates Angiopoietin-1 in endothelial developmental processes.

Asunto(s)

Clonación Molecular/métodos , Endotelio Vascular/citología , Glicoproteínas/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Neovascularización Fisiológica , Proteínas Tirosina Quinasas/fisiología , Proteínas/fisiología , Secuencia de Aminoácidos , Angiopoyetina 1 , Animales , Bovinos , División Celular , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/química , Glicoproteínas/genética , Corazón/embriología , Humanos , Hibridación in Situ , Ligandos , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Péptidos/inmunología , Fosfotirosina/metabolismo , Estructura Secundaria de Proteína , Ratas , Receptor TIE-2 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

Asunto(s)

Agrina/genética , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal/fisiología , Agrina/metabolismo , Animales , Eliminación de Gen , Expresión Génica/fisiología , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/fisiología , Unión Neuromuscular/química , Unión Neuromuscular/embriología , Unión Neuromuscular/fisiología , Fosforilación , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/fisiología , Tirosina/metabolismo

RESUMEN

The neurotrophins show a high degree of amino acid sequence homology, share similar solution properties, and display distinct but parallel functionalities. Here we report the crystallization and preliminary X-ray characterization of three neurotrophins: brain-derived neurotrophin, neurotrophin 3, and the heterodimer between brain-derived neurotrophin and neurotrophin 4. These findings are related to other published crystal parameters for neurotrophins, leading to the observation that, although crystal packing is highly variant, neurotrophins share common solubilities with respect to crystal growth.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/química , Cristalografía por Rayos X , Factores de Crecimiento Nervioso/química , Cristalización , Humanos , Neurotrofina 3 , Multimerización de Proteína , Proteínas Recombinantes/química

RESUMEN

The development and sustenance of specific neuronal populations in the peripheral and central nervous systems are controlled through the binding of neurotrophic factors to high-affinity cell surface receptors. The neurotrophins (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; and neurotrophin 4, NT4) are dimeric molecules which share approximately 50% sequence identity. The crystal structure of the murine NGF homodimer [McDonald et al. (1991) Nature 354, 411-414] indicated that the dimer interface corresponds to regions of high sequence conservation throughout the neurotrophin family. This potential compatibility was duly exploited for the production in vitro of noncovalent heterodimers between the different neurotrophins [Radziejewski, C., & Robinson, R.C. (1993) Biochemistry 32, 13350-13356; Jungbluth et al. (1994) Eur. J. Biochem. 221, 677-685]. Here, we report the X-ray structure at 2.3 A resolution of one such heterodimer, between human BDNF, and human NT3. The NGF, BDNF, and NT3 protomers share the same topology and are structurally equivalent in regions which contribute to the dimer interface in line with the propensity of the neurotrophins to form heterodimers. Analysis of the structure of regions of the BDNF/NT3 heterodimer involved in receptor specificity led us to conclude that heterodimer binding to p75 involves distant binding sites separately located on each protomer of the heterodimer. In contrast, heterodimer interactions with the trk receptors probably utilize hybrid binding sites comprised of residues contributed by both protomers in the heterodimer. The existence of such hybrid binding sites for the trk receptor provides an explanation for the lower activity of the BDNF/NT3 heterodimer in comparison to the homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Factores de Crecimiento Nervioso/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Factor Neurotrófico Derivado del Encéfalo , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Neurotrofina 3 , Estructura Secundaria de Proteína , Proteínas Recombinantes/química

RESUMEN

We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan family of receptor-like tyrosine kinases. These ligands correspond to protein S, a protease regulator that is a potent anticoagulant, and Gas6, a protein related to protein S but lacking any known function. Our results are reminiscent of recent findings that the procoagulant thrombin, a protease that drives clot formation by cleaving fibrinogen to form fibrin, also binds and activates intracellular signaling via a G protein-coupled cell surface receptor. Proteases and protease regulators that also activate specific cell surface receptors may serve to integrate coagulation with associated cellular responses required for tissue repair and growth, as well as to coordinate protease cascades and associated cellular responses in other systems, such as those involved in growth and remodeling of the nervous system.

Asunto(s)

Péptidos y Proteínas de Señalización Intercelular , Proteínas Oncogénicas/metabolismo , Proteína S/metabolismo , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Astrocitos/metabolismo , Northern Blotting , División Celular , Cromatografía de Afinidad , Regulación de la Expresión Génica , Ligandos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Oncogénicas/genética , Fosforilación , Unión Proteica , Proteína S/genética , Proteínas/genética , Proteínas Proto-Oncogénicas , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes de Fusión/metabolismo , Células de Schwann/metabolismo , Tirosina Quinasa del Receptor Axl

RESUMEN

We have determined that all four known members of the neurotrophin family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4), are capable of forming noncovalent heterodimers. The formation of these heterodimers was accomplished by homodimer subunit exchange promoted by treatment with guanidine hydrochloride, urea, low pH, or acetonitrile. In some cases (BDNF and mouse NGF; BDNF and NT-4), generation of the heterodimers was achieved by incubating homodimer mixtures in a neutral buffer at ambient temperature. The formation of heterodimers was in each case detected by nondenaturing gel electrophoresis at pH 7.4. High-performance cation-exchange chromatography was used to separate neurotrophin heterodimers from their parental homodimers. Heterodimers between BDNF and NT-3, BDNF and NT-4, and NT-3 and NT-4 are stable and show only a very small increase in homodimer content after 24 h of incubation at 37 degrees C. In contrast, heterodimers containing NGF subunits undergo gradual rearrangement to the homodimers. Our studies indicate that low pH, acetonitrile, and urea merely increase the neurotrophin subunit exchange rate and decrease the time needed to reach an equilibrium between a heterodimer and its two parental homodimers.

Asunto(s)

Factores de Crecimiento Nervioso/química , Proteínas del Tejido Nervioso/química , Animales , Factor Neurotrófico Derivado del Encéfalo , Cromatografía en Gel , Dicroismo Circular , Sustancias Macromoleculares , Ratones , Peso Molecular , Neurotrofina 3 , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes

RESUMEN

We have examined the molecular structure of the related neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) by physical methods, including gel filtration, velocity sedimentation, sedimentation equilibrium, urea gel electrophoresis, fluorescence spectroscopy, and far-ultraviolet circular dichroism. The results of these studies indicate that at physiologically relevant concentrations both recombinant proteins exist as tightly associated dimers. The dimers are stable even in 8 M solutions of urea. In solutions of guanidine hydrochloride, BDNF and NT-3 undergo slow unfolding between 3 and 5 M concentration of denaturant. Circular dichroism spectroscopy revealed approximately 70% beta-sheet and 20% beta-turn content in the native structure of both neurotrophic factors. In this respect, BDNF and NT-3 resemble other polypeptide growth factors whose receptors are also integral protein-tyrosine kinases.

Asunto(s)

Factores de Crecimiento Nervioso/química , Proteínas del Tejido Nervioso/química , Animales , Química Encefálica , Factor Neurotrófico Derivado del Encéfalo , Células CHO , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Dicroismo Circular , Cricetinae , Electroforesis , Calor , Humanos , Ratones , Neurotrofina 3 , Conformación Proteica , Desnaturalización Proteica , Espectrometría de Fluorescencia , Relación Estructura-Actividad

RESUMEN

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.

Asunto(s)

Transporte Axonal , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo , Células CHO , Cricetinae , Ganglios Espinales/metabolismo , Ganglios Simpáticos/metabolismo , Hipocampo/metabolismo , Radioisótopos de Yodo , Masculino , Ratones , Neuronas Motoras/metabolismo , Neurotrofina 3 , Prosencéfalo/metabolismo , Ratas , Ratas Endogámicas , Médula Espinal/metabolismo , Tálamo/metabolismo

RESUMEN

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

Asunto(s)

Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía en Gel , Factor Neurotrófico Ciliar , Clonación Molecular , ADN Recombinante/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes/genética , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Plásmidos , Ratas , Proteínas Recombinantes/genética

RESUMEN

A variety of findings seem to functionally link brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), while distinguishing both of these factors from the third member of the neurotrophin family, nerve growth factor (NGF). Here we demonstrate that all three of these neuronal survival molecules bind similarly to the low affinity NGF receptor, but that BDNF and NT-3, unlike NGF, do not act via the high affinity NGF receptor. However, both BDNF and NT-3, but not NGF, bind to full-length and truncated forms of a receptor-like tyrosine kinase, trkB, for which no ligand had previously been identified. In addition to binding BDNF and NT-3, trkB can mediate functional responses to both of these neurotrophins when it is expressed in PC12 cells, although BDNF appears to be the more effective ligand. Thus trkB encodes an essential component of a functional receptor for BDNF and NT-3, but not for NGF. Further evidence predicts the existence of additional functional receptors for the neurotrophins.

Asunto(s)

Glicoproteínas de Membrana/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/genética , Neoplasias de las Glándulas Suprarrenales , Animales , Unión Competitiva , Northern Blotting , Factor Neurotrófico Derivado del Encéfalo , Línea Celular , Reactivos de Enlaces Cruzados , Cinética , Glicoproteínas de Membrana/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neurotrofina 3 , Feocromocitoma , Unión Proteica , ARN/genética , ARN/aislamiento & purificación , Ratas , Receptores de Superficie Celular/metabolismo , Transfección

RESUMEN

L-leucyl-L-leucine methyl ester (LLME) is a lysosomotropic agent that in microMolar concentrations has been found to be selectively toxic to human and murine precursor and effector cytotoxic cells, irrespective of their surface membrane phenotype. We describe a new method of synthesis of LLME and evaluate the effects of this preparation on human lymphoid and hematopoietic progenitor cells. The new method of synthesis did not change the previously characterized activities of LLME. Consistent with previous observations, NK effectors, LAK precursors and effectors, and allospecific CTL (aCTL) effectors were completely ablated by treatment with 50-250 microM LLME, while the activities of helper T cells and B cells were preserved after treatments of up to 1000 microM LLME. The effects of LLME treatment on human marrow-derived erythroid, myeloid, and monocyte progenitors have not been previously described. We found that the growth of each of these committed precursors was reduced or eliminated following treatment with 100-250 microM LLME. Admixture of LLME-treated marrow with marrow depleted of T cells and other mature cellular elements resulted in increased growth of myeloid and erythroid colonies suggesting that cells that could provide colony-enhancing activities were preserved. In contrast to previous studies in humans, we found a minority of individuals to have aCTL precursors that were partially resistant to LLME. PBL from 10 of 15 individuals tested showed nearly complete ablation of aCTL precursors following treatment with 375 microM LLME. The remaining 5 individuals demonstrated significant aCTL precursor activity after identical treatment. The resistance to LLME was restricted to aCTL precursors, and neither increasing the dose of LLME nor prolonging the time of treatment completely overcame the resistance. The pattern of susceptibility (sensitive versus resistant) was found to be independent of the degree or type of HLA disparity between responder and stimulator. LLME-treated cultures with and without CTL activity contained a predominance of CD4+ T cells. However, in the subjects tested LLME-resistant aCTL was shown to be CD8+. In vitro priming of aCTL precursors from sensitive individuals did not consistently result in the development of resistance to LLME. These data indicate that further studies are needed to evaluate the effects of LLME on human stem cells and to determine the potential role of resistant aCTL precursors in GvHD prior to application of this technique as a form of selective T cell depletion in humans.

Asunto(s)

Citotoxicidad Inmunológica/efectos de los fármacos , Dipéptidos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Formación de Anticuerpos/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dipéptidos/síntesis química , Relación Dosis-Respuesta a Droga , Hematopoyesis/efectos de los fármacos , Humanos , Técnicas In Vitro , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos

RESUMEN

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.

Asunto(s)

Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas pp60(c-src) , Saccharomyces cerevisiae/crecimiento & desarrollo , Especificidad por Sustrato

RESUMEN

Using appropriately designed coenzyme analogues, new active sites can be introduced into naturally occurring enzymes by the chemical modification of specific residues. Catalytic activities very different from those of the corresponding native enzymes can be observed in the resulting semisynthetic enzymes. Covalent modification of the SH group of the active site residue Cys-25 of papain with flavins like 8-bromoacetyl-10-methylisoalloxazine converts the enzyme into a highly effective oxidoreductase. Thus, the catalytic versatility of existing enzymes can be enhanced through 'chemical mutation' of the active site.

Asunto(s)

Enzimas , Sitios de Unión , Catálisis , Fenómenos Químicos , Química , Coenzimas , Ingeniería Genética , Cinética , Mutación , Papaína , Moldes Genéticos