RESUMO
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Apoptose , Grânulos Citoplasmáticos/imunologia , Neutrófilos/imunologia , Neutrófilos/fisiologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Especificidade de Anticorpos , Antígenos CD/análise , Autoanticorpos , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Sobrevivência Celular , Grânulos Citoplasmáticos/ultraestrutura , Citometria de Fluxo , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Microscopia Imunoeletrônica , Neutrófilos/ultraestruturaRESUMO
Antibody-induced degradation and chemical cross-linking experiments have been carried out to assess the nature of the interaction between the two asialoglycoprotein-receptor polypeptides, H1 and H2, synthesized in HepG2 cells. Incubation of HepG2 cell monolayers with anti-H1 antibody caused a specific and equal loss of both H1 and H2 polypeptides. The same result was obtained with anti-H2 antibody. Control serum did not affect the level of H1 or H2 not did anti-H1 or anti-H2 antibodies affect the level of the transferrin receptor. The chemical cross-linking reagent, difluorodinitrobenzene, has been used to demonstrate that H1 can be cross-linked to H2 in HepG2 cell microsomal membranes. Dimer and trimer species with apparent molecular masses of 93 and 148 kD, respectively, were readily observed upon chemical cross-linking and some dimers and trimers were immunoreactive with both anti-H1 and anti-H2 antibodies. The putative trimer, possibly two H1 and one H2 molecules, is a minimum estimate of the true size of the asialoglycoprotein receptor in intact HepG2 cell, and it is possible that larger hetero-oligomeric forms of the receptor exist. The results of both types of experiments indicate that H1 and H2 form an oligomeric complex in HepG2 cells and thus, both polypeptides constitute the human asialoglycoprotein receptor.
Assuntos
Peptídeos/metabolismo , Receptores Imunológicos/metabolismo , Especificidade de Anticorpos , Receptor de Asialoglicoproteína , Carcinoma Hepatocelular , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoensaio , Neoplasias Hepáticas , Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific IgG molecules; the other chain was labeled with fluorescent monovalent Fab' fragments, and is lateral mobility was measured. In HepG2 cells, immobilization of either H1 or H2 led to an equal immobilization of the other, indicating that all the mobile H1 and H2 are in stable heterooligomers. In 1-7-1 cells, immobilization of H2 immobilized H1 to the same degree, but immobilization of H1 reduced the mobile fraction of H2 only by 2/3. Thus, in 1-7-1 cells all surface H1 molecules are associated with H2, but 1/3 of the H2 population is independent of H1. From these data and from measurements of the relative surface densities of H1 and H2, conclusions are drawn regarding the oligomeric structure and stoichiometry of the ASGP-R.
Assuntos
Receptores Imunológicos/química , Animais , Anticorpos , Receptor de Asialoglicoproteína , Linhagem Celular , Reagentes de Ligações Cruzadas , Imunofluorescência , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Receptores Imunológicos/metabolismo , TransfecçãoRESUMO
We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.
Assuntos
Endocitose , Endossomos/metabolismo , Insulina/metabolismo , Fígado/ultraestrutura , Receptor de Insulina/metabolismo , Animais , Endossomos/análise , Endossomos/ultraestrutura , Glucosídeos , Fígado/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
Studies were designed to evaluate the binding of binding of vitamin B12 to cell membrane preparations from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin B12 alone, binds to a single saturable binding site with an approximate Ka = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pretreating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the binding site includes a relatively exposed membrane protein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton X-100.
Assuntos
Proteínas Sanguíneas/metabolismo , Membrana Celular/metabolismo , Placenta/citologia , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Sítios de Ligação , Feminino , Humanos , Gravidez , Receptores de DrogaRESUMO
An acetone powder, prepared from the liver microsomes of vitamin K-deficient rats, retains an active vitamin K-dependent gamma-glutamyl carboxylase. While the basic requirements of the enzyme are similar to those of the carboxylase of either resuspended microsomes or detergent-solubilized microsomes, the acetone powder preparation reveals some additional properties of the carboxylase. Carboxylation of the synthetic pentapeptide substrate phenylalanylleucyl-glutamyl-glutamyl-valine can occur in the absence of nonionic detergent; however, when vitamin K hydroquinone drives the acetone powder carboxylation nonionic detergent is require for maximal activity. Experiments are described in which the acetone powder is incubated with the pentapeptide, pelleted by centrifugation, resuspended with fresh reactants, and incubated again. They suggest that the low V for the carboxylase, observed by all investigators, is, at least in part, not the result of irreversible enzyme inactivation nor depletion of reactants, but rather accumulation of a yet to be identified inhibitor(s). The acetone powder prepared from microsomes derived from livers of nutritionally normal cows contains vitamins vitamin K-dependent gamma-glutamyl carboxylase. This enzyme can be solubilized from the powder using Triton X-100 and could provided a large supply of starting material for enzyme purification.
Assuntos
Carbono-Carbono Ligases , Ligases/isolamento & purificação , Microssomos Hepáticos/enzimologia , Deficiência de Vitamina K/enzimologia , Acetona , Animais , Bovinos , Cinética , Ligases/metabolismo , Masculino , Oligopeptídeos , Pós , Ratos , Vitamina K/farmacologiaRESUMO
Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.
Assuntos
Ácidos Carboxílicos/metabolismo , Placenta/metabolismo , Vitamina K/metabolismo , Animais , Cálcio/metabolismo , Feminino , Humanos , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Gravidez , Ratos , Vitamina K/administração & dosagemRESUMO
Two related polypeptides, H1 and H2, comprise the human asialoglycoprotein receptor (ASGP-R). Stable lines of murine NIH 3T3 fibroblasts expressing H1 alone or H2 alone do not bind or internalize the ligand asialoorosomucoid (ASOR), which contains triantennary oligosaccharides. In contrast, cells expressing H1 and H2 together bind and degrade ASOR with properties indistinguishable from those of the ASGP-R in human hepatoma HepG2 cells. Whether or not H2 is coexpressed, H1 is synthesized as a 40-kDa precursor bearing high-mannose oligosaccharides, processed to its mature 46-kDa form, and transported to the cell surface. In cells expressing only H1, homodimers and -trimers of H1 are formed. In contrast, when expressed in 3T3 cells without H1, H2 is synthesized as its 43-kDa precursor, bearing high-mannose oligosaccharides, but is rapidly degraded. When H1 and H2 are coexpressed in the same cell, the H1 polypeptide "rescues" the H2 polypeptide; H2 is processed to its characteristic 50-kDa mature form and is transported to the surface. We conclude that the human ASGP-R is a multichain heterooligomer, probably a trimer of H1 molecules in noncovalent association with one, two, or three H2 molecules, and that the two polypeptides normally interact early in biosynthesis.
Assuntos
Assialoglicoproteínas , Receptores Imunológicos/metabolismo , Animais , Receptor de Asialoglicoproteína , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Camundongos , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Receptores Imunológicos/biossínteseRESUMO
The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself.
Assuntos
Biotina/metabolismo , Glutamatos/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Avidina/farmacologia , Biotina/antagonistas & inibidores , Biotina/deficiência , Fígado/enzimologia , Masculino , Tempo de Protrombina , Ratos , Vitamina KRESUMO
In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.
Assuntos
Insulina/farmacologia , Placenta/metabolismo , Proteínas Quinases/isolamento & purificação , Receptor de Insulina/isolamento & purificação , Feminino , Humanos , Cinética , Substâncias Macromoleculares , Gravidez , Proteínas Quinases/metabolismo , Receptor de Insulina/metabolismoRESUMO
An approach to isolate micromole quantities of tetraantennary oligosaccharides from human orosomucoid is presented. The N-linked oligosaccharides from 500 mg of the glycoprotein were released enzymatically, desialylated, and isolated free of protein using ion exchange chromatography. The pooled oligosaccharides were converted into oligosaccharide glycosylamines by reaction with ammonium bicarbonate then coupled to BOC-tyrosine to prepare tyrosinamide oligosaccharides. These were resolved on semipreparative RP-HPLC to recover micromole quantities of six purified tyrosinamide oligosaccharides. The oligosaccharide structures were elucidated by a combination of high-field proton NMR and matrix-assisted time of flight mass spectrometry and included biantennary, triantennary, monofucosylated triantennary, tetraantennary, monofucosylated tetraantennary, and a tetraantennary containing a single polylactosamine extension. Edman degradation was utilized to reverse the tyrosinamide oligosaccharide derivatization leading to the generation of reducing oligosaccharides. These were used to characterize the elution profile of asialyl orosomucoid oligosaccharides on high pH anion exchange chromatography. This application of tyrosinamide derivatization has allowed for the first time the complete resolution of the complex oligosaccharide mixture from orosomucoid on a semipreparative scale in a single chromatogram and provide the first NMR characterization of polylactosamine tetraantennary oligosaccharide from this substrate. This study demonstrates the broad utility of the tyrosinamide derivatization to develop oligosaccharide libraries useful for probing the biological functions of glycosylation.
Assuntos
Assialoglicoproteínas/química , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Orosomucoide/análogos & derivados , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Orosomucoide/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tirosina/análogos & derivadosRESUMO
We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically "nicked" in the beta subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor alpha subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.
Assuntos
Proteínas Quinases/metabolismo , Receptor de Insulina/metabolismo , Ditiotreitol/farmacologia , Etilmaleimida/farmacologia , Feminino , Histonas/metabolismo , Humanos , Substâncias Macromoleculares , Microssomos/análise , Elastase Pancreática/metabolismo , Fosforilação , Placenta/análise , GravidezRESUMO
Several mitogens elicit tyrosine-specific protein kinase activities. Although the physiological significance of this is unclear, the generality of these reactions implies that this may be an inherent feature of growth factor-growth factor receptor interactions. The observed mitogenic properties of the polypeptide insulin-like growth factor I (IGF-I) indicated that it might also stimulate such activity. We report here that IGF-I stimulates a tyrosine-specific protein kinase in a time- and dose-dependent fashion. The close correspondence between an approximate 50% effective dose (ED50) of phosphorylation and an approximate Kd for IGF-I binding leads us to conclude that a high-affinity IGF-I receptor, not the structurally similar insulin receptor, is the mediator of IGF-I stimulated kinase activity. Immunoprecipitation indicates that both the beta-subunit of the IGF-I receptor and the beta-subunit of the insulin receptor are targets for the IGF-I-stimulated protein kinase.
Assuntos
Insulina/farmacologia , Peptídeos/farmacologia , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/fisiologia , Somatomedinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Substâncias Macromoleculares , Peso Molecular , Fosforilação , Placenta , Gravidez , Proteínas Tirosina Quinases , Receptor de Insulina/fisiologia , Receptores de SomatomedinaRESUMO
The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expressing the two subunits in transfected fibroblast cell lines, it has been shown previously that the formation of a hetero-oligomeric complex is necessary for the transport of H2 to the plasma membrane and for high-affinity ligand binding. Here we show that subunit H1, when expressed in the absence of H2, is capable of internalization through coated pits and recycling. The kinetics of these processes are very similar to those of the H1-H2 complex. To study endocytosis in the absence of ligand binding, the cell surface was labeled at 4 degrees C with the 125I-iodinated impermeant reagent sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, the cells were incubated at 37 degrees C for different times and the amount of internalized receptor was determined by protease digestion of surface proteins and immunoprecipitation. Similarly, recycling of surface-labeled and then internalized receptor protein was studied by monitoring its reappearance on the surface in the presence of exogenous protease. Our results show that subunit H1 contains all the signals necessary for receptor endocytosis and recycling independent of ligand binding.
Assuntos
Endocitose , Receptores Imunológicos/metabolismo , Receptor de Asialoglicoproteína , Transporte Biológico , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Fibroblastos , Regulação da Expressão Gênica , Meia-Vida , Humanos , Imuno-Histoquímica , Peptídeo Hidrolases/metabolismo , Receptores Imunológicos/genéticaRESUMO
BACKGROUND: Alpha 2 mu-Globulin (A2), an 18.6 kD protein of hepatic origin, accumulates in the proximal tubule as an abundant, 15.5 kD cleavage product termed "A2-fragment" (A2-f). A2-f facilitates proximal tubule fatty acid oxidation, presumably by binding hydrophobic ligands. This requires some A2-f to enter the cytosol of the renal epithelial cell (REC). The localization of A2/A2-f in the proximal tubule cell was evaluated in this study. METHODS: Immunoblot analysis of renal cortical homogenates separated by differential centrifugation and quantitative immunoelectron microscopy (IEM) was performed to localize A2/A2-f using an affinity-purified antibody that detects both proteins. To evaluate A2 as a physiologically relevant ligand, the accumulation of A2-f in the female rat kidney (normally devoid of A2-f) was examined after the induction of hepatic A2 synthesis. Ligand binding, uptake, and degradation assays were used to assess A2 processing by RECs in vitro. RESULTS: Although A2 and A2-f were detected in the "lysosomal" fraction, only A2-f was found in the soluble protein fraction. IEM confirmed the presence of significant signal in the vesicular and lysosomal as well as the cytosolic compartments. In contrast, both beta 2 mu globulin (B2) and cathepsin B were restricted to endosomes. In the female rat, induction of hepatic A2 production resulted in A2-f accumulation in the renal cortex. In RECs in culture, uptake of A2 and B2 demonstrated nonsaturable, nondisplacable surface binding and similar uptake rates. Compared with B2, A2 was markedly resistant to degradation. CONCLUSIONS: A fraction of A2 escapes lysosomal degradation, permitting A2-f to accumulate in the cytosol of the proximal tubule epithelial cell. A2 may represent an unusual example of a physiologic protein capable of accumulating in a distant cell type.
Assuntos
alfa-Globulinas/metabolismo , Citosol/metabolismo , Túbulos Renais Proximais/metabolismo , Fígado/metabolismo , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Rim/metabolismo , Rim/ultraestrutura , Túbulos Renais Proximais/citologia , Masculino , Microscopia Imunoeletrônica , Gambás , Ratos , Distribuição Tecidual , Microglobulina beta-2/metabolismoRESUMO
The pentapeptide Phe-Leu-Glu-Glu-Leu, tritiated at the gamma carbon of each Glu residue, has been synthesized. In a system using microsomal preparations derived from rat liver, vitamin K-dependent tritium release from the L-Glu residues of this substrate can occur without the concurrent gamma-carboxylation of Glu. This tritium release reaction, which indicates cleavage of the gamma C-H bond, although easily uncoupled from CO2-dependent gamma C carboxylation, does require the reduced (hydroquinone) form of vitamin K and oxygen. The data argue against a concerted mechanism for the cleavage of the gamma C-H bond and carboxylation and against a mechanism in which the vitamin functions solely to transfer or activate CO2. Although the tritium release is related clearly to the oxidation of vitamin KH2, it is not yet established how the subsequent carboxylation proceeds. However, two carboxylation mechanisms compatible with the results are discussed.
Assuntos
Glutamatos/metabolismo , Peptídeos/metabolismo , Vitamina K/metabolismo , Animais , Ácidos Carboxílicos/metabolismo , Fenômenos Químicos , Química , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ratos , Trítio/metabolismoRESUMO
A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA library constructed in the expression vector lambda gt11. M13 vector sequence analysis yielded a deduced protein primary structure that was 89% homologous to the prototype alpha 1-inhibitor III (alpha 1I3) sequence presented in the preceding paper by Braciak et al. (Braciak, T. A., Northemann, W., Hudson, G. O., Shields, B. R., Gehring, M. R., and Fey, G. H. (1988) J. Biol. Chem. 263, 3999-4012) with regard to exact matches and 92% homologous when considering chemically conserved residues. The clone also possessed 100% homology to the putative bait region of a variant clone (pRLA1I3/27J) of alpha 1I3. Such sequence data demonstrates that the AF7 clone corresponds to a member of the family of variant alpha 1I3 mRNAs. Furthermore, this report presents the entire mRNA sequence corresponding to the 3'-half of alpha 1I3 variant 27J. We have utilized AF7 cDNA to study the expression of alpha 1I3 messenger RNA encoding this liver-specific glycoprotein under conditions known to alter hepatic gene expression. Our data reveal that alpha 1I3 mRNA expression is not only regulated during the acute-phase response but is also modulated in response to a variety of changing physiological conditions, most notably liver development. Steady state levels of mRNA were quantified using Northern blot techniques and laser densitometry. During acute phase response initiated by turpentine injection, the relative abundance of alpha 1I3 mRNA decreased 4-5-fold over a period of 24 h. Following partial hepatectomy, the regenerating liver expressed six-fold less alpha 1I3 mRNA than untreated liver after 24 h. This reduced level was maintained over a 2-day period. We have also demonstrated that alpha 1I3 mRNA expression is developmentally regulated. Fetal rat liver did not contain detectable concentrations of rat alpha 1I3 mRNA even as late as 4 days prior to birth. However, trace amounts were observed from birth until approximately 20 days of age when alpha 1I3 mRNA levels increased 10-fold to maximal adult quantities over the following 2 or 3 weeks. During the course of pregnancy, alpha 1I3 mRNA remained essentially constant until approximately 4 days prior to birth when a precipitous decline to 40% of the original level was noted. Subsequently, normal values were gradually restored over a 30-day postpartum period.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas de Fase Aguda , Fígado/metabolismo , Inibidores de Proteases/genética , RNA Mensageiro/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Genes , Variação Genética , Cinética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Gravidez , Ratos , Homologia de Sequência do Ácido Nucleico , alfa-Macroglobulinas/genéticaRESUMO
BACKGROUND: Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. However, its precise localization and function are not yet fully clarified. METHODS: To clone the rat homologue of nephrin, polymerase chain reaction (PCR) was employed. To elucidate the localization and expression of nephrin, immunohistological analysis with a specific antirat nephrin antibody, reverse transcription-PCR, and RNase protection assay were performed. RESULTS: Amino acid sequences of rat and human nephrin are highly homologous (82.2% identity). The domain structure of nephrin is also highly conserved between rats and humans. The rat nephrin was detected only in kidney glomeruli along glomerular capillary walls, and its localization was always identical to that of the anti-slit diaphragm monoclonal antibody (mAb) 5-1-6-recognized antigen in normal matured and fetal rat glomeruli and in the glomeruli of proteinuric states. The nephrin staining pattern was clearly distinguished from that of zonula occludens-1 (ZO-1), alpha3-integrin, or podocalyxin. mRNA expression for nephrin was first detected in the fetal rat kidneys at 18.5 embryonic days. Nephrin mRNA expression decreased just after injection of mAb 5-1-6 (47.4%) or puromycin aminonucleoside (51.2%), and the staining pattern of nephrin shifted from a linear to a granular pattern in both proteinuric states. CONCLUSIONS: Nephrin is localized in slit diaphragm in the matured glomeruli and is identical with mAb 5-1-6 antigen. Nephrin is involved in the development of proteinuria not only in mAb 5-1-6 nephropathy, but also in puromycin aminonucleoside nephropathy.
Assuntos
Glomérulos Renais/metabolismo , Proteínas/genética , Proteinúria/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Clonagem Molecular , Feto/metabolismo , Humanos , Glomérulos Renais/química , Glomérulos Renais/embriologia , Proteínas de Membrana , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas/análise , Proteinúria/etiologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the developing glomerulus by immunolocalization and metabolic labeling. The localization of p51 was compared with that of ZO-1, a component of the cytoplasmic face of the epithelial slit diaphragm, and with that of podocalyxin, and apical marker of the podocyte. p51 first became faintly, but clearly, detectable on the basal and lateral sides of the developing podocytes at the S-shaped body stage. Staining intensity increased with further maturation and was restricted to the visceral epithelial cells. On immunoelectron microscopy, the antigen was seen along the basal and lateral surfaces below occluding junction at the early capillary loop stage and later, with the interdigitation of foot processes, became concentrated in the slit pores. At no stage was p51 seen on the apical surface. p51 and ZO-1 were closely localized in the mature glomerulus but arrived at their final positions from opposite directions. p51 was on basal and podocalyxin was on apical sides of the glomerular epithelium from the S-shaped body stage onwards. Metabolic labeling studies showed that p51 is actively synthesized during initial glomerular development and that the rate of synthesis declines substantially with maturation. We conclude that p51 is primarily synthesized during the initial glomerular development, becomes concentrated in the slit pores of mature podocytes, and serves as a basal differentiation marker for podocytes.
Assuntos
Envelhecimento/metabolismo , Anticorpos Monoclonais/imunologia , Proteínas de Membrana/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/metabolismo , Endotélio/citologia , Endotélio/crescimento & desenvolvimento , Endotélio/metabolismo , Imunofluorescência , Glomérulos Renais/citologia , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/metabolismo , Microscopia Imunoeletrônica , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sialoglicoproteínas/metabolismo , Distribuição Tecidual , Proteína da Zônula de Oclusão-1RESUMO
Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier.