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1.
Synthesis of surface immunoglobulin E receptor in Xenopus oocytes by translation of mRNA from rat basophilic leukemia cells 总被引:7,自引:0,他引:7
Immunoglobulin E-binding activity was expressed in Xenopus oocytes injected with mRNA from rat basophilic leukemia cells which possess abundant immunoglobulin E (IgE) receptor. Such activity was demonstrated with intact oocytes by their binding of 125I-labeled mouse monoclonal IgE. Binding activity was specific as shown by the total inhibition of 125I-IgE binding by unlabeled IgE but not by unlabeled IgG1. The relevance of the IgE-binding activity to the IgE receptor was also supported by the absence of this activity in oocytes injected with mRNA from cells lacking surface IgE receptors. mRNA coding for the IgE-binding activity was enriched in fractions sedimenting at 13.5 S in sucrose density gradients. From oocytes injected with rat basophilic leukemia mRNA, two major polypeptides were isolated by affinity purification on IgE immunoadsorbent. One (Mr = 31,000) is equivalent in size to the previously identified "receptor-associated protein;" the other (Mr = 40,000) is speculated to be a partially glycosylated or unglycosylated form of the alpha subunit of the IgE receptor. The binding of IgE-coated fluorescent microspheres by oocytes injected with rat basophilic leukemia mRNA demonstrated the surface expression of the IgE-binding proteins. 相似文献
2.
Purification and characterization of the messenger RNA for the heavy chain of rat immunoglobulin E. 总被引:2,自引:0,他引:2
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The isolation and translational properties of rat immunoglobulin E (IgE) heavy chain mRNA are described. The mRNA has a sedimentation coefficient of approximately 18S, a chain length of about 2000 nucleotides and directs the synthesis in vitro of a polypeptide of 65000 molecular weight in an mRNA-dependent rabbit reticulocyte lysate. Inclusion of dog pancreatic microsomes in the cell-free translation system resulted in a heavy chain product of about 75000 molecular weight, presumably as a consequence of glycosylation in vitro. This species co-migrated in an SDS polyacrylamide gel with mature IgE heavy chain. Substantial purification of heavy chain mRNA was achieved by denaturing sucrose gradient centrifugation and agarose gel electrophoresis. 相似文献
3.
The fate of IgE bound to rat basophilic leukemia cells. IV. Functional association between the receptors for IgE 总被引:1,自引:0,他引:1
K Furuichi J Rivera T Triche C Isersky 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(3):1766-1773
Rat basophilic leukemia cells (RBL-2H3) have receptors for immunoglobulin E (IgE) and immunoglobulin G (IgG). These receptors for IgE mediate the endocytosis of chemically or immunochemically cross-linked IgE but not monomeric IgE. However, unoccupied receptors were endocytosed with cross-linked IgE. To further assess the degree and specificity of the observed coendocytosis, we exposed cells carrying monomeric rat IgE and monomeric mouse IgE anti-DNP to a DNP-protein conjugate. We found that up to 30% of the surface-bound monomeric rat IgE redistributed at 0 to 4 degrees C and was then internalized at 37 degrees C with the immunochemically cross-linked mouse IgE. To assess the specificity of the coendocytosis, we exposed cells carrying monomeric rat IgE to immunochemically cross-linked mouse IgG. We found that the binding, patching, and endocytosis of cross-linked mouse IgG had no effect on the monomerically bound rat IgE. The rate of coendocytosis was the same as the rate of endocytosis (t 1/2 3 to 5 min). The extent of coendocytosis depended on the extent of endocytosis but was relatively insensitive to changes in the ratio between mouse and rat IgE over a broad range. These results indicate that some of the receptors for IgE are associated in a specific fashion. 相似文献
4.
We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release. 相似文献
5.
Tomoko Adachi Makio Mogi Minoru Harada Kohichi Kojima 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,668(2):327-332
We examined the suitability of an immunoaffinity membrane [rabbit IgG specific for rat immunoglobulin E (IgE) immobilized on a cellulose membrane] for removing IgE from rat blood passed through a simple extracorporeal circulatory system. To determine the concentration of IgE in the blood, we also developed a highly sensitive chemiluminescence enzyme immunoassay for rat IgE. The IgE levels in the outlet blood from the immunoaffinity membrane module decreased to 30% of the initial concentration within 30 min. 相似文献
6.
Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine. 总被引:2,自引:0,他引:2
S L Erlandsen C B Rodning C Montero J A Parsons E A Lewis I D Wilson 《The journal of histochemistry and cytochemistry》1976,24(10):1085-1092
Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A. 相似文献
7.
Molecular weight of heavy chains of immunoglobulin G typical of cancer is studied immunoglobulin and may be responsible for manifestation of certain anomalous acid and peptide composition of this protein heavy chains as compared with immunoglobulin G in blood serum of healthy people. Immunochemical methods helped detecting an antigenic determinant (or determinants) which is arranged in the heavy chains of the studied immunoglobulin and may be responsible for manifestation of certain anomalous properties of cancer-typical immunoglobulin G molecules. A set of bromo-cyanogenic fragments differing from the spectrum of these fragments in the heavy chains of normal immunoglobulin G is formed following a specific chemical effect of bromo-cyanogen on the heavy chains of immunoglobulin G typical of cancer. Essential differences are found in dancyl-fingerprints of the heavy chains of the compared proteins. Everything mentioned is a result of changes in the primary structure of the heavy chains of immunoglobulin G typical of cancer. 相似文献
8.
S K Kops R E Ratzlaff R Meade G M Iverson P W Askenase 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(12):4515-4524
Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells. 相似文献
9.
A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of 125I-labeled protein A (125I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of 125I-PA. The degree of inhibition of 125I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays. 相似文献
10.
One‐step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice
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Jon M Steichen Sven Kratochvil Eleonora Melzi Johan Arnold Stephanie K Dougan Lin Wu Kathrin H Kirsch Usha Nair William R Schief Facundo D Batista 《The EMBO journal》2018,37(18)
Here, we describe a one‐step, in vivo CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in mice. We produced knock‐in (KI) mice wherein a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease‐induced double‐stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double‐stranded breaks are subsequently repaired via homology‐directed repair by a plasmid‐borne template containing the pre‐arranged human immunoglobulin heavy chain. To validate our knock‐in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B‐cell development and performing single B‐cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock‐in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases. 相似文献
11.
In this work, we have used Xenopus oocyte maturation as a read-out for examining the ability of the neu tyrosine kinase (p185neu) to participate with the epidermal growth factor (EGF) receptor in a common signal transduction pathway. We find that unlike the case for the EGF receptor, which elicits EGF-dependent maturation of these oocytes as reflected by their germinal vesicle breakdown (GVBD), neither the normal neu tyrosine kinase (p185val664) nor the oncogenic form of neu (p185glu664) are able to effectively trigger this maturation event. However, expression of p185glu664 causes a specific and significant promotion of the progesterone-induced GVBD, reducing the half-time for this maturation even from approximately 9 h to approximately 5 h. Stimulation of the progesterone-induced GVBD did not occur following the expression of a kinase-deficient p185neu protein (in which a lysine residue at position 758 was changed to alanine). Essentially identical results were obtained when the mRNAs coding for fusion proteins comprised of the extracellular domain of the receptor for immunoglobulin E (IgE), and the membrane-spanning and tyrosine kinase domains of normal or oncogenic p185neu (designated IgER/p185val664 and IgER/p185glu664, respectively), were injected into oocytes. Antigen-induced crosslinking of IgER/p185val164 proteins expressed in oocytes caused a reduction in the half-time for the progesterone-stimulated GVBD from approximately 9 h to approximately 7 h. Thus, the aggregation of the membrane-spanning and/or tyrosine kinase domains of p185val664 partially mimics the effects of the oncogenic forms of p185neu. Overall, the results of these studies suggest that the activation of the p185neu tyrosine kinase by a point mutation within its membrane-spanning helix, or an aggregation event, can result in the facilitation of oocyte maturation events that are elicited by other factors (e.g. progesterone). However, the activated p185neu tyrosine kinases are not able to mimic the EGF-stimulated EGF receptor tyrosine kinase in triggering oocyte maturation, which suggests that the EGF receptor and the p185neu tyrosine kinase do not input into identical signal transduction pathways in these cells. 相似文献
12.
In earlier studies, the specific adherence of normal rat macrophages to Schistosoma mansoni schistosomula, followed by macrophage cytotoxicity against the larvae, was shown to be induced by incubation of the macrophages with serum from infected rats containing complexes of IgE antibody and circulating schistosome antigens. By the use of a chromium-51 release assay, it is pointed out that this cytotoxic process is a two-step phenomenon. The first step, i.e., activation of normal unstimulated macrophages induced by incubation of the cell with IgE complexes in immune rat serum, is a nonspecific mechanism which may also be elicited by various other macrophage activators. The second step, i.e., immune adherence and cytotoxicity of activated macrophages against S. mansoni schistosomula, is a specific process which imperatively needs the presence of S. mansoni IgE immune complexes. Aggregated myeloma IgE does not activate adherent peritoneal cells into cytotoxic effector cells unless the further participation of these specific IgE immune complexes is provided. The necessary preincubation of macrophages with immune rat serum before adding schistosomula accounts for the inefficiency of the incubation of the target itself with serum to elicit macrophage cytotoxicity. Serum dilution also appears as a critical factor since immune rat serum is inefficient when diluted more than . Aggregated rat IgG neither induces macrophage activation nor inhibits the activation by IgE immune complexes. Though the binding of IgE to the macrophage appears to be isotype specific, homologous immune complexes of IgE antibody and schistosome antigens are required to induce killing of S. mansoni larvae. The possible mechanism of this new model of macrophage activation and cytotoxicity is discussed. 相似文献
13.
Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts 总被引:23,自引:15,他引:8
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We have used a model system to explore the importance of long-range lateral diffusion of membrane proteins in specific membrane-membrane adhesion. Single, cell-size phospholipid vesicles containing a dinitrophenyl (DNP)-lipid hapten were maneuvered into contact with rat basophilic leukemia (RBL) cells carrying fluorescent anti-DNP IgE in their cell-surface Fc epsilon receptors. Upon cell-vesicle contact the antibody molecules underwent a marked lateral redistribution, accumulating at the site of contact and becoming significantly depleted from noncontacting membrane. As assayed with a micropipette suction method, there was a time-dependent increase in the strength of cell-vesicle adhesion. This development of adhesion paralleled the kinetics of accumulation of the adhesion-mediating antibody molecules at the zone of membrane-membrane contact. Both adhesion and redistribution were absolutely dependent upon a specific interaction of the IgE with the hapten: No redistribution occurred when vesicles lacking the DNP hapten were pushed against IgE-armed RBL cells, and on cells bearing a 1:1 mixture of nonimmune rat IgE and anti-DNP mouse IgE, only the latter underwent redistribution. Vesicles containing DNP-lipids bound to RBL cells carrying anti-DNP IgE but not to cells carrying nonimmune rat IgE. Measurable nonspecific binding did not develop even after 15 min of pushing DNP-bearing vesicles against RBL cells sensitized with nonimmune IgE. Neither redistribution nor adhesion was blocked by metabolic poisons such as NaN3 and NaF. Both redistribution and adhesion occurred in plasma membrane blebs previously shown to lack cytoskeletal filaments. The above observations are consistent with contact-induced redistribution of the IgE being a result of passive diffusion-mediated trapping rather than active cellular responses. Thus, long-range diffusion of specific proteins can in some cases contribute to the formation of stable adhesion between membranes. 相似文献
14.
Extracellular release of rat eosinophil peroxidase (EPO) I. Role of anaphylactic immunoglobulins 总被引:6,自引:0,他引:6
J Khalife M Capron J M Grzych H Bazin A Capron 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(3):1968-1974
The release of intracellular peroxidase (EPO) was investigated in order to evaluate rat eosinophil activation by various immunoglobulin (Ig) isotypes. After successive incubations with purified rat IgG1, IgG2a, IgG2b, IgG2c, IgE, or IgM and their respective anti-Ig antisera, eosinophils released significant amounts of EPO (up to 26% of the intracellular content) only in the case of Ig with anaphylactic activities (IgG2a and IgE). Other classes and subclasses were unable to induce EPO exocytosis. Selective depletion and reconstitution experiments suggested that mast cells were not required in this process. Similar levels of EPO could be released after interaction of eosinophils with antigen-antibody complexes (IgG2a monoclonal antibody and Schistosoma mansoni antigen) immobilized on nonphagocytosable surfaces. These results indicate that EPO exocytosis can be obtained after cell activation with specific antibodies, and that this mechanism is independent of phagocytosis. A kinetic study of eosinophils from S. mansoni-infected rats revealed that IgG2a and IgE cytophilic antibodies induced EPO release after incubation with either specific antisera or specific antigen, which suggests the in vivo relevance of such findings. The present work underlines the parallelism of interaction of anaphylactic-type Ig with eosinophils and with mast cells. Moreover, EPO release seems to represent an interesting marker of eosinophil activation, because close relationships were established between the present findings and previous work on the effector function of rat eosinophils. 相似文献
15.
Covalent cross-linking of subunits of the receptor for immunoglobulin E induced by immunoprecipitation 总被引:3,自引:0,他引:3
The receptor on rat basophilic leukemia and related normal cells that binds monomeric immunoglobulin E (IgE) with high affinity contains four polypeptide chains: alpha (to which the IgE binds), beta, and a disulfide-linked dimer of gamma chains. In this study, we have analyzed a further component variably seen when the purified receptors are analyzed on polyacrylamide gels. This component has an apparent Mr of approximately 43 000 and, after treatment with reducing agents, yields one beta and two gamma chains. This complex is generated by immunoprecipitation of preparations totally lacking in it. This novel in vitro phenomenon has provided additional information about the structure of the receptor. Its possible relationship to in vivo aggregation that triggers degranulation of the cells is of interest. 相似文献
16.
D Brassart E Kolodziejczyk D Granato A Woltz M Pavillard F Perotti L G Frigeri F T Liu Y Borel J R Neeser 《European journal of biochemistry》1992,203(3):393-399
A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy. 相似文献
17.
Hanashiro K Tokeshi Y Nakasone T Sunagawa M Nakamura M Kosugi T 《Mediators of inflammation》2001,10(4):217-221
BACKGROUND: Although the levels of immunoglobulin E (IgE) in the circulating blood are often elevated in patients with allergic diseases, such levels cannot always be considered as pathognomonic signs of allergy. The induction of allergic reactions in the tissue was inferred to be related to the amount of IgE passing through the vascular wall. AIMS: We attempted to clarify which compartment, the intravascular or extravascular, plays an important role in the regulation of the turnover of rat IgE. METHODS: The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. RESULTS: The transfer rate constants from the central to tissue compartment (Kct) were larger than those from the tissue to central compartment (Ktc) irrespective of the sensitized state. The value of the distribution volume of the tissue compartment (Vt) was larger than that of the distribution volume of the central compartment (Vc) irrespective of the sensitized state. CONCLUSIONS: These Findings suggest that the short half-life of rat IgE in the circulation could be attributable to the distribution of IgE from the intravascular to the extravascular compartment. 相似文献
18.
《International journal for parasitology》1996,26(7):787-791
Investigation of restriction fragment length polymorphisms (RFLPs) associated with immunoglobulin E (IgE) and cytokine genes in the sheep genome revealed polymorphisms in the IgE constant heavy chain, interferon γ and interleukin 4 genes. No polymorphisms were found in interleukin 1β or tumour necrosis factor α. PstI and BamHI RFLPs in the IgE gene showed differences in frequency between animals selected for resistance or susceptibility to fleece rot and blowfly strike. 相似文献
19.
Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors. 总被引:17,自引:0,他引:17
Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release. 相似文献
20.
Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E 总被引:5,自引:0,他引:5
The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites. 相似文献