Mast cells and basophils are immunologically activated by aggregation of IgE molecules bound to the Fc.epsilon.RI with multivalent antigen. Cell response can also be induced by directly cross-linking the Fc.epsilon.RI, for example, with anti-receptor antibodies. Clustering of the Fc.epsilon.RI on mast cells and basophils by either IgE and polyvalent antigen or directly by specific monoclonal antibodies, initiates a cascade of biochemical processes coupling to the cells secretory response. These include: (i) the activation of receptor-associated protein tyrosine kinases (Eiseman and Bolen, 1992) and phosphatases (Hampe and Pecht, 1994) causing a transient increase in tyrosine phosphorylation of several cellular proteins (Benhamou et al., 1990 and 1992); (ii) an increase in phosphoinositides hydrolysis (Beaven et al., 1984a) resulting from PLC.gamma.1 activation (Li et al., 1992); and (iii) the rise in the intracellular concentration of free calcium ions (Beaven et al., 1984b). The final response to this stimulus is the secretion of granule-stored mediators and the de novo synthesis and secretion of mediators of inflammation and the allergic response, including histamine, serotonin, arachidonic acid metabolites, like leukotrienes (Ortega et al., 1989), prostaglandins, and several cytokines (Bradding et al., 1993; Galli et al., 1991).
Several mast cell membrane components different from known Fc.epsilon.RI subunits have been identified on the rat mucosal type mast cell line RBL-2H3, mainly by specific monoclonal antibodies (mAb), and shown to modulate the Fc.epsilon.RI-mediated secretory response. G63, a mAb that binds a membranal glycoprotein named MAFA, for mast cell function-associated antigen (Ortega and Pecht, 1988), was shown to inhibit both the Fc.epsilon.RI-induced signalling cascade upstream to PLC.gamma.1 activation (e.g. phosphatidylinositide hydrolysis products and transient rise in the cytoplasmic concentration of free Ca.sup.2+ ions), and the culminating secretion of the cells' granule contents (Ortega and Pecht, 1988). mAb G63 inhibitory effect required MAFA clustering, and was not due to interference with IgE- Fc.epsilon.RI interactions. Still, cross-linking of Fc.epsilon.RI-IgE complexes by multivalent antigen also led to co-clustering of the MAFA with the aggregated Fc.epsilon.RI and in the enhancement of its internalization (Ortega et al., 1991).
The MAFA has been identified as a glycoprotein with a MW of 28 to 40 kDa distinct from any known Fc.epsilon.RI subunit by immunoprecipitation with mAb G63 and reducing SDS-PAGE. When the SDS-PAGE was run under non-reducing conditions, the observed pattern was different: a component with an apparent MW of 60-82 kDa was detected in addition to the above described 28 to 40 kDa band, suggesting that the MAFA is a disulfide-linked dimer composed of subunits of similar size (Ortega and Pecht, 1988).
Following the discovery of MAFA, the present inventors sought to isolate and sequence the gene encoding this protein. However, the standard procedures for isolating and cloning desired genes proved to be unsuitable for isolating and cloning the gene encoding MAFA (hereinafter mafa). Originally, peptides thought to be derived from MAFA were obtained and sequenced using standard peptide sequencing procedures, and used to prepare oligonucleotides for use as probes to isolate the mafa sequence in RBL cells. However, this proved to be unsuccessful and it was found that the original peptides were not MAFA-derived. Further, as MAFA is not an enzyme and there is no known natural ligand which binds to MAFA, it was also not possible to carry out usual standard cloning and subsequent screening procedures. Thus, after considerable experimental effort, the successful cloning, isolation and sequencing of the mafa sequence, in accordance with the present invention, was achieved by so-called "eukaryotic expression-cloning", a procedure detailed herein below in which from a large number of transfected mammalian cells a single suitable transfected clone was identified and isolated using emulsion autoradiography (Gearing et al., 1989) by virtue of its expressing the MAFA correctly as a surface-bound protein. This isolation was here for the first time achieved by using as ligand a radiolabeled monoclonal antibody (G63) specific for MAFA. Moreover, only upon the successful isolation and sequencing of the mafa sequence, in accordance with the present invention, was it possible to determine precisely the molecular weight of the protein (both the monomeric and homodimeric forms), the glycosylation sites on the protein, and also to perform an accurate sequence determination of the cDNA encoding it and deduce its amino acid sequence, thus allowing a comparative analysis of the MAFA sequence with the sequences of other known mammalian proteins.
There has been a long-felt need to provide a highly-specific modulator of inflammation and/or allergic reactions (i.e. reagents that would block the secretory response of MC and basophils to the immunological stimulus). Most known substances used to treat inflammation and allergic reactions, e.g. anti-histamines, treat the consequences of the secretion process rather than inhibit it. In addition, they also have undesirable side-effects. MAFA is a highly-specific modulator of the initial stages of the cellular processes leading to inflammation and allergic reactions. The characterization of mafa affords a tool for screening for potential MAFA ligands, which ligands alone or in combination with MAFA can be used to prevent inflammatory and allergic reactions. The DNA molecule encoding MAFA provides a basis for large-scale production of this potentially pharmaceutically-important protein, in its native form or in a modified soluble form thereof.
USPTO Backgrounds
USPTO Backgrounds