Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-23
2001-01-09
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C436S501000
Reexamination Certificate
active
06171803
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to DNA segments encoding the &agr;, &bgr;, and &ggr; subunits of the high affinity receptor for immunoglobulin E (IgE). The invention further relates to a method of producing the receptor by expressing DNA encoding its &agr;, &bgr;, and &ggr; subunits in a host cell simultaneously.
2. Related Art
Receptors that bind the Fc region of immunoglobulins (“Fc receptors”) mediate immunoglobulin transport across membranes, stimulate a variety of cellular activities induced by antigen-antibody complexes, and possibly regulate the biosynthesis of antibodies. Three of the receptors (the receptor for polymeric immunoglobulin (Mostov et al. (1984) Nature (London) 308:37-43), the Fc receptors on macrophages and lymphocytes (Ravetch et al. (1986) Science 234:718-725), and the high affinity Fc, receptor on mast cells and basophils (Kinet et al. (1987) Biochemistry 26:4605-4610; Shimizu et al. (1988) Proc. Natl. Acad. Sci USA 85:1907-1911; Kochan et al. (1988) Nucleic Acids Res. 16:3584) share a common feature: their immunoglobulin-binding portion contains two or more immunoglobulin-like domains.
The high affinity IgE receptor Fc
&egr;
RI is responsible for initiating the allergic response. Binding of allergen to receptor-bound IgE leads to cell activation and the release of mediators (such as histamine) responsible for the manifestations of allergy. This receptor is a tetrameric complex &agr;&bgr;&ggr;
2
which is found on the surface of mast cells and basophils. The &agr; and &bgr; subunits have not been detected in other hematopoietic cells although the &ggr; chains of FceRI are found in macrophages, NK cells and T cells where they associate with the low affinity receptor for IgG (Fc&ggr;RIII) or with the T cell antigen receptor.
The genes for &agr; and &ggr;, both have been localized on human (Le Coniat, 1990) and mouse chromosome 1. (Huppi, 1988; Kinet et al. 1987; Kochan et al. 1988; Shimizu et al. 1988; Ra et al. 1989.) The gene for mouse &bgr; has been localized on mouse chromosome 19 and is believed to be a single gene (Huppi, 1989). The structures of the &agr; gene in the rat (Tepler, 1989) and of the &ggr; gene (Kuster, 1990), but not of the &bgr; gene have been characterized in the human.
The receptor with high affinity for the IgE Fc
&egr;
RI is found exclusively on mast cells, basophils, Langerhans cells, and related cells. Aggregation of IgE occupied Fc
&egr;
RI by antigen triggers both the release of preformed mediators such as histamine and serotonin, as well as stimulation of the synthesis of leukotrienes. It is the release of these mediators that results in the allergic condition.
The most thoroughly characterized Fc
&egr;
FI is that of the rat basophilic leukemia (FEL) cell line. It consists of three different subunits: (1) a 40-50 Kilodalton (Kd) glycoprotein alpha chain which contains the binding site for IgE, (2) a single 33 Kd beta chain and (3) two 7-9 Kd disulfide linked gamma chains (H. Metzger et al., Ann. Rev. Immunol. 4:419-470 (1986).
Complementary DNA (cDNA) for the rat &agr; subunit has been isolated (J.-P. Kinet et al., Biochemistry 26:4605-4610 (1987)). However, previously there has been no disclosure of the isolation and characterization of the &bgr; and &ggr; subunits nor has it been possible to express IgE-binding by transfected cells (J.-P. Kinet et al., Biochemistry 26:4605-4610 (1987); A. Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911 (1988)).
Molecular cloning of some of the subunits in rodents and humans has permitted the reconstitution of surface expressed receptor complexes by trasfection. One of the surprising findings from these studies was the differential requirement for surface expression among different species. Cotransfection of the three chains, &agr;, &bgr; and &ggr; is required to promote efficient surface expression of the rat (Blank, 1989) or mouse receptor (Ra., 1989). By contrast, some surface expression of the human &agr;&ggr; complex can be achieved by cotransfecting &agr; and &ggr; alone suggesting that &bgr; may not be necessary (Miller, 1989). This result and previous inability to clone the gene for the human &bgr; subunit raised the possibility that human beta might not exist and that &agr;&ggr; complexes might exist naturally in human cells.
The high affinity IgE receptor Fc
&egr;
RI is a tetrameric hetero-oligomer composed of an &agr; chain, a &bgr; chain and two disulfide-linked &ggr; chains (chains and subunits will be used interchangeably herein). The &bgr; chain contains four transmembrane (TM) segments and long cytoplasmic domains which are thought to play an important role in intracellular signalling. It was very difficult to determine whether a human beta subunit even existed, and if so, to isolate its gene. The present invention has overcome these difficulties and surprisingly provided cDNA clones for the human &bgr; subunit of Fc
&egr;
RI.
The invention still further provides a method of producing the complete human Fc
&egr;
RI receptor, and for inhibiting formation of the receptor or its function, by inhibiting the &bgr; subunit.
SUMMARY OF THE INVENTION
It is an aspect of this invention to provide nucleic acid segments encoding Fc
&egr;
RI subunits.
It is an aspect of this invention to provide nucleic acid sequences encoding the &agr;, &bgr;, and &ggr; subunits of Rc
&egr;
RI. In particular, this invention relates to DNA sequences. An aspect of the present invention is the structural characterization and the sequence of the complete human &bgr; gene and cDNA. Successful cloning of the human beta was not expected and was fraught with failures. Attempts to clone the human beta by simply using a rodent beta probe to screen various cDNA libraries failed to isolate a cDNA clone encoding human beta. Only a very short fragment (153 bp) with homology to rodent beta was isolated. However because this fragment may have been a portion of a beta-like molecule such as CD20, known to be homologous to beta in that region, PCR techniques were used to clone the human beta by using the information from the rodent beta sequence. However, although the homologies between human and rodent beta were 69% in the coding region, that was not sufficient for a PCR reaction. Human beta isolated by this method also failed.
The existence of human beta was questioned because human beta was believed not necessary for expression of the alpha-gamma complex. Studies of gene transfer indicated that the transfer by transfection of the three genes for alpha, beta and gamma was necessary for the expression of the rat and mouse receptor. However, transfection of human alpha and gamma was sufficient to promote the surface expression of the human receptor in fibroblasts suggesting that the human beta was not necessary for the surface expression of the human receptor. That result raised the interesting question of the existence of human beta.
Human beta was not necessary for the function of the alpha-gamma complex. Transfection of the cytoplasmic tail of gamma is sufficient for cell activation. Several groups made the observation that the cytoplasmic domain in the gamma chain was sufficient to mediate a number of biochemical signals leading to cell activation. These signals include tyrosine kinase activation, hydrolysis of phosphoinositides, calcium mobilization, production of IL2 in T cells, degranulation of mast cells and cell killing. It was demonstrated that the cytoplasmic domains of gamma contain a motif of 10-12 amino-acid residues responsible for cell activation. This motif is sufficient to trigger many different signals in different cells. It is transferable, and seemed to be interchangeable. Again these findings raised the question of the existence of human beta. If the gamma chain is sufficient for cell activation, perhaps there was no need for a beta.
The inability to clone the human beta or even to detect transcripts for human beta in human cells (by using rat or mouse probes) also raised the question of the existence of human beta.
Cloning required invent
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
The United States Government as represented by the Department of
Ulm John
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