RESUMEN
Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, alpha-smooth muscle actin phenotype, and expression of beta-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-beta1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, platelet- and macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease. (J Histochem Cytochem 47:533-543, 1999)
Asunto(s)
Fibronectinas/biosíntesis , Glomerulonefritis Membranoproliferativa/metabolismo , Trombospondina 1/biosíntesis , Animales , División Celular , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/genética , Glomerulonefritis Membranoproliferativa/patología , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Hibridación in Situ , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Trombospondina 1/genética , Factores de TiempoRESUMEN
Rat proximal tubular epithelial cells derived from Wistar-Kyoto and spontaneously hypertensive rats were grown to confluency on semipermeable tissue culture inserts, and the plasminogen system of these cells was analyzed using enzyme assays, Western analysis, zymography, and reverse transcriptase-polymerase chain reaction. The tubular epithelial cells are capable of activating exogenous plasminogen to plasmin by endogenous plasminogen activators. The cells produce tissue-plasminogen activator, urokinase-plasminogen activator, plasminogen activator inhibitor-1, and urokinase-plasminogen activator receptor. These cells also produce the Heymann nephritis autoantigen, gp330 (megalin), and an associated protein of 45 kd (RAP). Incubation with transforming growth factor-beta 1 resulted in a decrease in plasminogen activation, primarily because of an increase in plasminogen activator inhibitor-1 RNA and protein and a decrease in u-PA RNA as noted by quantitative reverse transcriptase-polymerase chain reaction, Western analysis, and zymography. Incubation of these cells with tumor necrosis factor-alpha resulted in an increase in plasminogen activating ability, presumably through an increase in urokinase. Gp330 and the associated 45-kd protein (RAP) RNA were decreased in cells treated with tumor necrosis factor-alpha. The data presented indicates that these transformed proximal tubular epithelial cells may be used to study changes that may occur during Heymann nephritis with respect to the plasminogen system and the autoantigen gp330.
Asunto(s)
Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Plasminógeno/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Glomerulonefritis/etiología , Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Complejo Antigénico de Nefritis de Heymann , Túbulos Renales Proximales/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Reacción en Cadena de la Polimerasa , ARN/genética , ARN/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Rat glomerular epithelial cells were grown to confluency on semipermeable tissue culture inserts and the plasminogen system of these cells was analyzed using enzyme assays, Western analysis, zymography, and reverse transcriptase-PCR. The glomerular epithelial cells are capable of activating exogenous plasminogen to plasmin by endogenous plasminogen activators. The cells produce both tissue-plasminogen activator and urokinase-plasminogen activator with urokinase being the prominent activator. Both activators are present primarily on the basolateral side of the cells with urokinase found primarily at the cell surface presumably bound to its receptor and tissue-plasminogen activator found primarily in the matrix secreted by the cells on the semipermeable insert. The cells also produce plasminogen activator inhibitor-1 and urokinase-plasminogen activator receptor. Inhibition of plasminogen activation occurred with plasminogen activator inhibitor-1, anti-catalytic anti-tissue-plasminogen activator antibody, epsilon-aminocaproic acid, which inhibits the binding of plasminogen through its lysine binding sites, and amiloride, which specifically inhibits urokinase.
Asunto(s)
Fibrinolisina/análisis , Glomérulos Renales/metabolismo , Plasminógeno/análisis , Animales , Secuencia de Bases , Células Cultivadas , Epitelio/metabolismo , Datos de Secuencia Molecular , Plasminógeno/metabolismo , Reacción en Cadena de la Polimerasa , RatasRESUMEN
The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.
Asunto(s)
Autoantígenos/química , Glomerulonefritis/inmunología , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Animales , ADN Complementario/química , Complejo Antigénico de Nefritis de Heymann , Glomérulos Renales/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Ratas , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The plasminogen (Plg) system on rat yolk sac carcinoma (L2) cells was characterized by zymography, Western and immunoprecipitation analysis, reverse-transcriptase polymerase chain reaction, binding, and activity assays. The L2 cells produced tissue Plg activator but not urokinase Plg activator and contained RNA for Plg activator inhibitor 1, but Plg activator inhibitor 1 was not detectable by zymography or Western analysis and contained the receptor for urokinase Plg activator. Plg bound to the cells in a saturable manner when plasmin inhibitors were present with a dissociation constant of 1.34 +/- 0.18 x 10(-6) M and 1.54 +/- 0.25 x 10(7) sites/cell. Immunoprecipitation analysis showed that Plg was binding to gp330, a known Plg receptor. Once bound to the L2 cells, Plg was activated by tissue Plg activator to plasmin in a time- and concentration-dependent manner. Under saturating Plg conditions, most of the plasmin produced was released into the medium. Inhibition of plasmin activation occurred when Plg activator inhibitor 1, anticatalytic tissue Plg activator antibody, or Heymann nephritis autoantibody was present.
Asunto(s)
Tumor del Seno Endodérmico/metabolismo , Plasminógeno/metabolismo , Animales , Secuencia de Bases , Tumor del Seno Endodérmico/patología , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/metabolismo , Complejo Antigénico de Nefritis de Heymann , Glicoproteínas de Membrana/metabolismo , Sondas Moleculares/genética , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/farmacología , Activadores Plasminogénicos/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
Previous results have shown that the autoantibody eluted from the glomeruli of rats with active Heymann nephritis contain a population of antibodies not only to the putative autoantigen of the disease, gp330, but also to plasminogen. Since gp330 has been shown to serve as a receptor for plasminogen, we have analyzed the effects of autoantibody on plasminogen-binding to gp330 and activation of plasminogen to plasmin by urokinase. Autoantibody does not inhibit the binding of plasminogen to gp330. The binding of autoantibody to plasminogen was shown to be very specific for the compact conformation of glu-plasminogen. The change in the conformation of plasminogen when its lysine-binding sites are occupied or after conversion to plasmin results in a significant decrease in autoantibody-binding. The most significant effect of autoantibody on this system is the inhibition of plasminogen activation to plasmin by urokinase. The binding of autoantibody to plasminogen acts as a competitive inhibitor of the reaction by apparently blocking access of urokinase to plasminogen's activation site. These results indicate that autoantibody obtained from the immune deposits in the glomeruli of rats with active Heymann nephritis does not inhibit the binding of plasminogen to gp330 but does significantly alter the urokinase catalyzed activation of plasminogen to plasmin.
Asunto(s)
Autoanticuerpos/farmacología , Glomerulonefritis/inmunología , Glicoproteínas de Membrana/inmunología , Plasminógeno/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Autoanticuerpos/inmunología , Autoanticuerpos/aislamiento & purificación , Unión Competitiva , Fibrinolisina , Complejo Antigénico de Nefritis de Heymann , Glomérulos Renales/inmunología , Cinética , Glicoproteínas de Membrana/metabolismo , Plasminógeno/metabolismo , RatasRESUMEN
Previously, we have shown that gp330, the putative autoantigen of Heymann nephritis which is located on the glomerular and tubular epithelial cells, is a receptor for plasminogen. During our analysis of the function of gp330, we also observed by means of Western analysis the binding of gp330 to a 45-kDa protein. This 45-kDa protein was obtained from extracts of rat kidney and purified to apparent homogeneity. Enzyme-linked immunosorbent assay analysis indicate that gp330 can bind to the 45-kDa protein. In fact, both the 45-kDa protein and plasminogen can bind to gp330 simultaneously, indicating that gp330 has two separate sites for each respective protein. No inhibition of binding of either ligand to gp330 is observed when both ligands were simultaneously incubated with immobilized gp330. A rabbit polyclonal antibody raised against this 45-kDa protein was used to screen a rat kidney lambda gt11 expression library. Sequence analysis of the isolated clones revealed that this protein is similar to the human alpha 2-macroglobulin receptor-related protein and murine heparin-binding protein-44. It is also identical to a previously reported, supposedly pathogenic, domain of gp330. However, our results show that the cDNAs encode for a 45-kDa protein, which is not a part of gp330 but a separate protein which binds to gp330.
Asunto(s)
Autoantígenos/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/aislamiento & purificación , Secuencia de Bases , Northern Blotting , Western Blotting , ADN/genética , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Complejo Antigénico de Nefritis de Heymann , Riñón/inmunología , Glomérulos Renales/inmunología , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Poli A/genética , Poli A/aislamiento & purificación , Unión Proteica , Proteínas/genética , Proteínas/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Previously, we demonstrated that the Heymann nephritis autoantigen, gp330, can serve as a receptor site for plasminogen. This binding was not significantly inhibited by the lysine analogue epsilon-amino caproic acid (EACA), indicating that plasminogen binding was not just through lysine binding sites as suggested for other plasminogen binding sites. We now report that once plasminogen is bound to gp330, it can be converted to its active form of plasmin by urokinase. This conversion of plasminogen to plasmin proceeds at a faster rate when plasminogen is first prebound to gp330. Although there is a proportional increase in the Vmax of the urokinase-catalyzed reaction with increasing gp330 concentrations, no change in Km was observed. Once activated, plasmin remains bound to gp330 in an active state capable of cleaving the chromogenic tripeptide, S-2251. The binding of plasmin to gp330 did not significantly change its enzymatic activity; however, gp330 did have a stabilizing effect on plasmin activity at 37 degrees C. While bound to gp330, plasmin is protected from inactivation by its natural inhibitor alpha 2-antiplasmin. The binding of plasmin to gp330 as analyzed by ELISA was shown to be time dependent, reversible, saturable, and specific for gp330. Inhibition of binding of both plasminogen and plasmin to gp330 by benzamidine was similar, although EACA inhibited the binding of plasmin to gp330 slightly more than the binding of plasminogen to gp330. These results indicate that the binding of plasminogen to gp330 serves as an effective means of increasing the rate of plasmin production on the glomerular and tubular epithelial cell surface while protecting the active plasmin from natural inhibitors.
Asunto(s)
Fibrinolisina/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Activación Enzimática , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/farmacología , Complejo Antigénico de Nefritis de Heymann , Técnicas In Vitro , Cinética , Unión Proteica , Ratas , alfa-Macroglobulinas/farmacologíaRESUMEN
Previous results have demonstrated the binding of a 76- and 80-kDa serum protein to the Heymann nephritis autoantigen, gp330. This 76-kDa serum protein was purified by column chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit polyclonal antibody for the serum protein was produced and used to screen a rat liver cDNA expression library. Sequence analysis of an isolated clone identified the serum protein as plasminogen. Plasminogen was isolated from rat serum by standard techniques, and the binding of plasminogen to gp330 was confirmed by Western analysis. Enzyme-linked immunosorbent assay results demonstrated a time-dependent, saturable, and specifically inhibitable binding of plasminogen to gp330. There was no significant difference in the binding of the two carbohydrate forms of plasminogen to gp330. Plasminogen binding to gp330 could be completely inhibited by the addition of exogenous gp330. This binding could also be partially inhibited by benzamidine but only slightly by the lysine analogue, epsilon-aminocaproic acid. However, even a combination of these two inhibitors could not completely block the binding of plasminogen to gp330 indicating that gp330 may be binding to plasminogen through some other unknown interactions. These results demonstrate that gp330 is a receptor site for plasminogen.
Asunto(s)
Autoantígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasminógeno/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , Biblioteca de Genes , Complejo Antigénico de Nefritis de Heymann , Cinética , Hígado/fisiología , Datos de Secuencia Molecular , Peso Molecular , Plasminógeno/genética , Plasminógeno/inmunología , Plasminógeno/aislamiento & purificación , Ratas , Receptores de Superficie Celular/inmunología , Receptores del Activador de Plasminógeno Tipo UroquinasaRESUMEN
We have used affinity purified antibody, which reacts with the renal antigen gp600, to examine the immunoreactivity of pulmonary cells in the quick frozen lung tissue of fetal rats. On immunoelectron microscopy, we found that the reaction was specifically localized to the luminal surfaces of Clara cell and, as previously reported, to alveolar type II cell (ATII) membranes. No immunoreaction was seen on or in type I cells, endothelial cells or any other lung or blood cell. These studies suggest that the anti-gp600 immunoselection can be used as a marker to identify and quantify Clara and ATII cells in rat lung tissue.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Feto/patología , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Pulmón/patología , Glicoproteínas de Membrana/inmunología , Alveolos Pulmonares/patología , Animales , Membrana Celular/inmunología , Complejo Antigénico de Nefritis de Heymann , Técnicas In Vitro , Pulmón/inmunología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/ultraestructura , RatasRESUMEN
Because of the similarities between Heymann nephritis of rat and human membranous glomerulonephritis, we searched for a protein in human kidney similar to that of the putative autoantigen of Heymann nephritis, gp330. Human gp330 was isolated by a purification scheme identical to that used for the isolation of rat gp330. The isolated human gp330 was compared with rat gp330 by peptide mapping and was found to be very similar in structure. Studies of immunologic reactivity of the rat antigen-induced Heymann nephritis nephritogenic autoantibody to human gp330 indicated that some of the rat antigen epitopes were shared with its human counterpart. The human gp330 was capable of eliciting on active Heymann nephritis response in rats when used as an immunogen. The nephritogenic autoantibody eluted from these rat kidney glomeruli showed equal binding to both rat and human antigens. Finally, a polyclonal antibody to human gp330 was able to induce passive Heymann nephritis in rats. These results demonstrate that there is a gp330 protein in human kidney that is very similar to the putative autoantigen of Heymann nephritis.
Asunto(s)
Autoantígenos/química , Glomerulonefritis/metabolismo , Glicoproteínas/aislamiento & purificación , Riñón/metabolismo , Glicoproteínas de Membrana/aislamiento & purificación , Animales , Autoanticuerpos/inmunología , Reacciones Cruzadas , Glomerulonefritis/inmunología , Glicoproteínas/química , Glicoproteínas/inmunología , Complejo Antigénico de Nefritis de Heymann , Humanos , Sueros Inmunes/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Peso Molecular , Mapeo Peptídico , Ratas , Ratas EndogámicasRESUMEN
Two antibody probes were used to characterize the putative renal antigens of HgCl2-induced antiglomerular basement membrane renal disease in Brown Norway (BN) rat. The first probe was the linear immunofluorescence imparting, in vivo bound, nephritogenic antiglomerular-basement-membrane autoantibody (anti-GBM-Ab). The second probe was a rat monoclonal antibody to the B subunit of laminin that was obtained from fusion of spleen cells of HgCl2 injected BN rat. By enzyme-linked immunosorbent assay (ELISA) the anti-GBM-Ab reacted with laminin, type IV collagen, collagenase-resistant noncollagenous portion of glomerular basement membrane (GBM), saline soluble proteins of kidney cortex homogenate and fibronectin. Western blot analysis of laminin indicated that the reactive epitopes detected by both probes were on the B chain subunit but not the A subunit. In nonreduced collagenase-digested GBM the epitopes were present on 27 kD and 42 to 48 kD polypeptides. A similar pattern was seen on collagenase-digested human GBM. On rat and human GBM the patterns obtained with rat autoantibody and autoantibody from a patient with Goodpasture syndrome were similar, suggesting that some of the in vivo bound anti-GBM autoantibodies in HgCl2-induced disease in rat are directed against epitopes which are similar to the Goodpasture antigen of human. Reactive epitopes were also detected on saline soluble proteins of kidney cortex homogenate with the predominant antigen being a 31 kD polypeptide. In the saline soluble proteins the reactive polypeptides including the major 31 kD polypeptide did not originate from laminin, type IV collagen, or the collagenase-resistant noncollagenous part of GBM. The precise structural origin of soluble proteins was not defined.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos/inmunología , Autoantígenos/inmunología , Colágeno Tipo IV , Glomerulonefritis/inmunología , Riñón/inmunología , Cloruro de Mercurio/toxicidad , Animales , Anticuerpos Monoclonales , Autoanticuerpos , Autoantígenos/análisis , Membrana Basal/inmunología , Western Blotting , Colágeno/análisis , Colágeno/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Glomerulonefritis/inducido químicamente , Laminina/inmunología , Masculino , Ratas , Ratas Endogámicas BNRESUMEN
Heymann nephritis [model of membranous glomerulonephropathy (MGN)] kidneys (n = 20) with proteinuria were transplanted into unilaterally nephrectomized normal syngeneic Lewis recipient rats to study the course of established MGN lesion in normal milieu. Concurrent with the loss of C3 staining from the MGN lesion at 2 to 4 wk after transplantation, the proteinuria decreased from the transplanted kidney (p less than 0.02). Thereafter, from 2 to 28 wk no further decrease was noted in proteinuria which stabilized at a lower but still abnormal level. No appreciable decrease in IgG deposits in MGN lesion was noted up to 12 wk but at 28 wk the deposits had decreased significantly (p less than 0.005). However, the deposits did not resolve completely even at 40 wk. The results indicate that in MGN one component of proteinuria is due to ongoing activation of C with the deposition of new antibody and the other is perhaps due to structural damage to glomerular filter. The former appears reversible and the later irreversible. Although the resolution of IgG deposits in the lesion is very slow significant improvement can occur with time (several months).
Asunto(s)
Glomerulonefritis Membranosa/etiología , Glomerulonefritis/etiología , Trasplante de Riñón , Proteinuria/etiología , Animales , Femenino , Estudios de Seguimiento , Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Glomerulonefritis Membranosa/inmunología , Glomerulonefritis Membranosa/metabolismo , Supervivencia de Injerto , Inmunoglobulina G/metabolismo , Glomérulos Renales/análisis , Complicaciones Posoperatorias/etiología , Proteinuria/inmunología , Proteinuria/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
Based on our previous finding in lung parenchyma of high concentration of the shared epitopes of gp600, a well characterized kidney glycoprotein, we attempted to identify the anatomic site of these epitopes and characterize them biochemically. Affinity-purified polyclonal anti-gp600 antibody was used as the probe. Immunocytochemically in lung on light and electron microscopy the probe reacted exclusively with type II pneumocytes and no other lung cell. The reaction was also demonstrated on freshly isolated and cultured type II pneumocytes. Both approaches showed the reaction to localize on the cell membrane of type II pneumocytes. Immunoprecipitation of radiolabeled type II pneumocyte cell membranes identified two 270- to 290-kDa polypeptides as the reactive proteins. We conclude that the reactive epitopes for anti-gp600 in lung parenchyma are exclusively localized on type II pneumocytes and have a Mr of approximately 270 to 290 kDa and that anti-gp600 may be used as a specific immunologic marker for the type II pneumocytes. Finally, it is possible that the differences in the molecular forms of the cross-reactive proteins in lung and kidney identified in this report are the reason for the known non-nephritogenicity of rat lung for the induction of Heymann nephritis in rat.
Asunto(s)
Antígenos de Superficie/análisis , Alveolos Pulmonares/análisis , Animales , Antígenos de Superficie/inmunología , Antígenos de Superficie/aislamiento & purificación , Membrana Celular/análisis , Reacciones Cruzadas , Citoplasma/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Pruebas de Precipitina , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/ultraestructura , Conejos , Ratas , Ratas EndogámicasRESUMEN
Earlier studies have localized the Heymann nephritis (HN) autoantigen (gp330) in the coated pits of the plasma membrane and multivesicular bodies of the glomerular epithelial cell. Because of these locations in the glomerular epithelial cells, it has been suggested that the HN Ag may be a receptor. The aim of our study was to search for a ligand which can bind the HN autoantigen. Normal rat serum was subjected to SDS-PAGE under reducing and non-reducing conditions followed by Western analysis of the separated polypeptides. A reaction was revealed directly by autoradiography using 125I labeled HN autoantigen as a probe and indirectly by enzyme immunodetection using unlabeled nephritogenic autoantibody (anti-gp330) eluted from glomeruli of diseased rats followed by biotinylated rabbit anti-rat IgG avidin-peroxidase complex. A polypeptide of 76 kDa Mr was identified under non-reducing conditions as a serum protein reacting with the HN autoantigen. Reactivity of the 76-kDa polypeptide was lost when serum was electrophoresed under reducing conditions. Direct binding of the 76-kDa polypeptide obtained from serum to the HN autoantigen obtained from kidney suggests that the 76-kDa polypeptide may be a ligand for the autoantigen. This is the first documentation of a possible ligand for the HN autoantigen. Not only does this polypeptide bind to the HN autoantigen but it also shows direct binding with the nephritogenic autoantibody eluted from glomerular deposits. This characteristic of the 76-kDa polypeptide indicates that this serum protein may potentially play a role in the development of the glomerular lesion of active HN. Further analysis of this serum component should assist in understanding the normal function of the HN autoantigen.
Asunto(s)
Autoantígenos/análisis , Proteínas Sanguíneas/metabolismo , Glomerulonefritis/sangre , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Autoanticuerpos/análisis , Autoantígenos/inmunología , Sitios de Unión de Anticuerpos , Proteínas Sanguíneas/aislamiento & purificación , Glomerulonefritis/inmunología , Complejo Antigénico de Nefritis de Heymann , Glomérulos Renales/metabolismo , Glicoproteínas de Membrana/inmunología , Peso Molecular , Ratas , Ratas Endogámicas Lew , Receptores Inmunológicos/aislamiento & purificaciónRESUMEN
The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase (OPRT; orotidine-5'-phosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine-5'-monophosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase; EC 4.1.1.23). In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in lambda gt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases.
Asunto(s)
Carboxiliasas/genética , Clonación Molecular , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Pentosiltransferasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/enzimología , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
Conflicting results of previous electron microscopy studies and concerns about the validity of immunoperoxidase technique employed in those studies to accurately localize endogenous IgG in rat glomerular basement membrane (GBM) prompted us to use other techniques to answer the following question: Does endogenous IgG permeate the matrix of GBM? Immunofluorescence, radioimmunoassay (RIA), isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunodetection on Western blots were used to detect endogenous IgG in GBM. Direct immunofluorescence of normal frozen rat kidney sections prepared from in vivo perfused kidney showed endogenous IgG in a linear pattern of staining in the GBM. RIA for rat IgG found the IgG content of collagenase-solubilized GBM to be 0.48% of the dry weight. Immunodetection for rat IgG on Western blots of SDS-PAGE-separated GBM demonstrated endogenous IgG in purified collagenase-solubilized GBM. IgG was detected as an intact molecule with covalently linked light and heavy chains and not as small immunoreactive catabolic fragments. Isoelectric focusing followed by immunodetection on Western blot showed that part of the endogenous IgG in GBM was anionic. The results clearly show that under normal conditions, endogenous IgG can permeate into the collagen matrix of GBM in rat and that some of this IgG is more anionic than the IgG in serum. These findings may assist in understanding the transit of autoantibodies to subepithelial glomerular antigens located beneath the matrix of GBM in membranous glomerulonephropathy.
Asunto(s)
Inmunoglobulina G/metabolismo , Glomérulos Renales/ultraestructura , Animales , Membrana Basal/metabolismo , Técnica del Anticuerpo Fluorescente , Focalización Isoeléctrica , Masculino , Microscopía Electrónica , Radioinmunoensayo , Ratas , Ratas Endogámicas LewRESUMEN
Three different phenotypes have been characterized in HeLa cells that have been selected for resistance to pyrazofurin, a potent inhibitor of the de novo pyrimidine biosynthetic enzyme UMP synthase. All of the resistant cell lines had a coordinate increase in UMP synthase activity, UMP synthase-specific mRNA, and UMP synthase gene sequences. In one of the resistant cell lines, the amplification of the UMP synthase gene is associated with a stable phenotype. There is no decrease in UMP synthase gene copy number or UMP synthase activity when these cells are grown for over six months in the absence of pyrazofurin. Another resistant cell line that has a higher level of gene amplification when grown in the presence of pyrazofurin loses its elevated UMP synthase activity and amplified DNA sequences with growth in the absence of the drug. A third cell line that possessed a moderate level of UMP synthase gene amplification is tenfold more resistant to pyrazofurin than the cell line with the highest level of amplification. The extraordinary level of resistance is due to a decreased level of activity for the enzyme adenosine kinase that is required for the conversion of pyrazofurin to its inhibitory monophosphate form.
Asunto(s)
Carboxiliasas/genética , Células HeLa/enzimología , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Pentosiltransferasa/genética , Adenosina Quinasa/deficiencia , Amidas , Biotransformación , ADN de Neoplasias/análisis , Resistencia a Medicamentos , Amplificación de Genes , Células HeLa/efectos de los fármacos , Humanos , Complejos Multienzimáticos/antagonistas & inhibidores , Orotato Fosforribosiltransferasa/antagonistas & inhibidores , Orotidina-5'-Fosfato Descarboxilasa/análisis , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Fenotipo , Pirazoles , ARN Mensajero/análisis , ARN Neoplásico/análisis , Ribonucleósidos/metabolismo , Ribonucleósidos/farmacología , RibosaRESUMEN
The levels of UMP synthase protein and mRNA are increased in rat hepatoma cells that have acquired resistance to pyrazofurin, a potent inhibitor of pyrimidine biosynthesis. A cDNA plasmid library was prepared from partially purified poly(A)+ mRNA isolated from the resistant cell line. Recombinant plasmids with inserts complementary to UMP synthase mRNA were selected by differential hybridization with cDNA prepared from wild type and resistant cell mRNA and analysis of hybrid-selected mRNA by in vitro translation reactions. One plasmid, pUMPS-2, contains a 850-base pair insert and was used to analyze UMP synthase gene sequences in the wild type and resistant cell lines. Blot hybridization of restricted genomic DNA demonstrated amplification of the UMP synthase gene in the resistant cells. The number of UMP synthase genes is increased 15-fold as determined by a modified dot hybridization procedure. Previous studies have shown that the resistant cells have a 16-fold increase in UMP synthase mRNA but a 40-fold increase in synthase activity (Suttle, D.P. (1983) J. Biol. Chem. 258, 7707-7713). To further investigate this discrepancy between the amount of increase in DNA and mRNA versus the increase in enzyme activity, we have determined the relative rate of synthesis and degradation of UMP synthase. The rate of synthesis was 13-fold faster in the resistant cells. The degradation rate was not significantly different between the two cell lines. These data indicate that gene amplification is the major factor contributing to the enzyme overproduction in the pyrazofurin-resistant cells.