WO1995027734A1 - A dna molecule encoding a mast cell function-associated antigen (mafa) - Google Patents
A dna molecule encoding a mast cell function-associated antigen (mafa) Download PDFInfo
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- WO1995027734A1 WO1995027734A1 PCT/US1995/004258 US9504258W WO9527734A1 WO 1995027734 A1 WO1995027734 A1 WO 1995027734A1 US 9504258 W US9504258 W US 9504258W WO 9527734 A1 WO9527734 A1 WO 9527734A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions
- the present invention is generally in the field of inflammation and allergy and relates to substances which may be useful in controlling the cellular processes which result in inflammation or allergic reactions. More specifically, the present invention concerns a new isolated DNA molecule 0 encoding a protein, the mast cell function-associated antigen (hereinafter MAFA) , which is capable of modulating the response to the type I Fc ⁇ receptor for IgE (hereinafter Fc ⁇ Rl) , which is present on the surface of mast cells, basophils, eosinophils and Langerhans cells, and 5 which activates these cells.
- MAFA the mast cell function-associated antigen
- Fc ⁇ Rl type I Fc ⁇ receptor for IgE
- the present invention also concerns recombinant expression vectors containing the MAFA- encoding DNA molecule and host cells transformed by the vectors which are capable of expressing biologically active MAFA.
- the invention also provides a method for screening t - ) potential ligands of MAFA, which ligands alone or in combination
- Mast cells and basophils are immunologically activated by aggregation of IgE molecules bound to the Fc ⁇ RI with multivalent antigen.
- Cell response can also be induced by directly cross-linking the Fc ⁇ RI, for example, with anti-receptor antibodies.
- 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).
- mAb monoclonal antibodies
- 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 RI-induced signalling cascade upstream to PLC7I activation (e.g.
- the MAFA has been identified as a glycoprotein with a MW of 28 to 40 kDa distinct from any known Fc ⁇ RI subunit by immunoprecipitation with mAb G63 and reducing SDS-PAGE.
- 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) .
- the present inventors sought to isolate and sequence the gene encoding this protein.
- the successful cloning, isolation and sequencing of the mafa sequence 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.
- 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.
- the isolation, in accordance with the present invention, of the DNA molecule encoding MAFA provides a means for obtaining one or more soluble form(s) of MAFA, which may be used to screen for potential ligands, e.g. saccharides, lipopolysaccharides, proteins, glycoproteins, and glycopeptides that bind specifically to MAFA.
- potential ligands e.g. saccharides, lipopolysaccharides, proteins, glycoproteins, and glycopeptides that bind specifically to MAFA.
- preparation of such soluble form(s) of MAFA also provides a means for generating additional monoclonal antibodies having specificity for different epitopes (from that of mAb G63) of MAFA.
- the present invention provides an isolated DNA sequence encoding a mammalian mast cell function- associated antigen (MAFA) . Further, the present invention also provides an isolated DNA sequence encoding a polypeptide product of prokaryotic or eukaryotic host expression, said product having all or part of the primary structural conformation of a mammalian MAFA and preferably having the binding specificity of MAFA as determined in a binding assay with an anti-MAFA monoclonal antibody (mAb) , for example, mAb G63 (G63) .
- mAb monoclonal antibody
- the present invention comprises a DNA sequence selected from the group consisting of : (a) DNA molecules comprising a nucleotide sequence encoding native mammalian
- the invention provides the cDNA sequence depicted in Fig. 5 which encodes native MAFA and cDNA sequences which encode a soluble form of MAFA having said binding specificity of MAFA and an amino acid sequence which is contained within the sequence of amino acid residues 1-188 depicted in Fig. 5.
- the present invention also provides recombinant expression vectors comprising any of the above noted DNA sequences of the invention; prokaryotic or eukaryotic host cells transfected by the vectors and capable of expressing native MAFA or a soluble form of MAFA having said binding specificity of MAFA; a process for preparing MAFA or said soluble form thereof comprising culturing suitable host cells under conditions promoting expression; and a mammalian MAFA and soluble forms thereof produced by the process.
- the present invention provides a method for screening potential ligands of MAFA which comprises :
- a suitable vector modifying said mafa sequence by addition, deletion or substitution mutagenesis of more than one codon to generate a modified-MAFA-encoding vector; expressing said modified-MAFA-encoding vector in a suitable host cell to obtain a modified-MAFA protein that is a soluble form of MAFA, or converting said modified-MAFA into a soluble form of MAFA if necessary; assaying the binding specificity of said soluble MAFA by determining its ability to bind to a monoclonal antibody (mAb) specific to MAFA; and selecting a soluble form of MAFA capable of binding said mAb.
- mAb monoclonal antibody
- the mafa sequence can be mutagenized using standard methods (Sambrook et al., 1989) to delete or change the amino-terminal non- cleavable signal sequence in order to produce a secreted, soluble MAFA.
- soluble MAFA can be obtained using commercially available systems for inserting all or part of the mafa sequence into an appropriate vector able to direct its expression as part of a fusion protein (containing a tag for affinity purification) in the host of choice (prokaryotic, insect cells or mammalian cell lines) .
- the present invention also provides ligands specific to MAFA obtained by the above screening method of the invention, and compositions containing one or more of said ligands alone or in combination with the MAFA which may be used to prevent inflammatory and allergic reactions.
- Fig. 1 depicts electrophoretic analysis of the native and N-deglycosylated MAFA.
- a lysate derived from 108 surface-radioiodinated RBL-2H3 cells was incubated with mAb G63-coated beads, which were then washed and incubated with N-glycosidase F (lanes l and 3) or with buffer alone (lanes 2 and 4), as described in Experimental Procedures.
- the immuno-precipitates were eluted by boiling in SDS-PAGE sample buffer and analyzed on a 10 to 17.5% polyacrylamide gel under reducing (lanes 3 and 4) or non-reducing conditions (lanes 1 and 2) . The gel was then dried and autoradiographed.
- Fig. 2 shows two dimensional tryptic peptide mapping of the monomeric and dimeric forms of N- deglycosylated MAFA.
- the 18 and 40 kDa forms were separated by SDS-PAGE as described for Fig. l, visualized by autoradiography, and excised.
- the two gel pieces were then submitted in parallel reactions to radioiodination with the chloramine T method and to tryptic proteolysis as described in Experimental Procedures.
- the peptide fragments that eluted from the gel pieces were separated on cellulose- coated plates by electrophoresis in one dimension and chromatography in the other. The plates were then autoradiographed.
- Fig. 1 shows two dimensional tryptic peptide mapping of the monomeric and dimeric forms of N- deglycosylated MAFA.
- the 18 and 40 kDa forms were separated by SDS-PAGE as described for Fig. l, visualized by autoradiography, and excised.
- the two gel pieces were then submitted in parallel reactions to
- 3A depicts flow cytometric analysis of COS-7 cells transfected with the MAFA cloned cDNA, and stained with mAb G63.
- 106 transfected COS-7 cells were incubated for 30 minutes at room temperature in the presence (shadowed histogram) or absence (empty histogram) of 0.5 mg/ml biotinylated mAb G63, washed, and further incubated for another 30 minutes at 37(C in the presence of 25 -
- the fluorescence associated with 5000 cells of each sample was analyzed with a Becton Dickinson FacScan flow cytometer, and the derived histograms are shown overlaid.
- Fig. 3B shows immunoprecipi a ion and SDS-PAGE analysis of the protein encoded by the cloned cDNA.
- the immunoprecipitates were eluted by boiling in SDS-PAGE sample buffer and separated on a 12.5% polyacrylamide gel under reducing or non-reducing conditions. The gel was then dried and autoradiographed.
- Fig. 4A shows N-deglycosylation of the protein encoded by the cloned MAFA cDNA.
- 107 transfected COS-7 cells were surface-radioiodinated, and the derived lysate was incubated with mAb G63-coated beads. The beads were then washed and incubated with N-glycosidase F as described in Experimental Procedures.
- a sample of MAFA immunoprecipitated from surface-radioiodinated RBL-2H3 cells was N-deglycosylated following the same procedure. The immunoprecipitates were then subjected to reducing SDS-PAGE on a 15% polyacrylamide gel, and electrotransferred to a nitrocellulose membrane. The membrane was then autoradiographed.
- Fig. 4B shows endoproteinase Lys-C digestion of both the protein encoded by the cloned cDNA and the MAFA.
- the nitrocellulose strips containing the 20 kDa polypeptide cores visualized in Fig. 4 A were excised from the membrane and incubated with endo-proteinase Lys-C as described in
- Fig. 5 depicts the nucleotide sequence of the cloned MAFA cDNA.
- the deduced amino acid sequence of the MAFA is shown in the one-letter code. Bases and residues are numbered on the left.
- the predicted transmembrane domain is underlined, the putative N-glycosylation sites are boxed and the cysteins are circled.
- the TGA stop codon is marked with an asterisk and the polyadenylation signal is overlined.
- Fig. 6 shows sequence homology between the MAFA and members of the C-type lectin family.
- the MAFA carboxy terminal 114 amino acids are aligned with the carbohydrate recognition region (CRD) of selected C-type lectins with which the highest degree of homology is shared. Fully conserved residues are boxed and the motifs CYYF and WIGL are shadowed. The first and last amino acid of each displayed sequence is numbered in bold and the total length of the respective polypeptide is given in brackets.
- m-LECI mouse asialoglycoprotein receptor 2
- m-Fce2 mouse Fc(RII; h-CD69: human CD69
- m-NKll mouse natural killer cell antigen NKR-P1.
- sequences were aligned with the PILEUP program in the GCG sequence analysis software.
- the nucleotide sequence of the cloned MAFA cDNA will appear in the EMBL, DDBJ, and GenBank Nucleotide Sequence Databases under the accession number X79812.
- Fig. 7 shows phosphoamino acid analysis of the 32P-labeled MAFA.
- PVDF stripes containing 20 kDa radiolabeled MAFA were excised, washed and heated to 110°C in 6 N HCl for one hour. The HCl was then removed by evaporation and the hydrolysates were resuspended in 5 ⁇ l pH 3.5 buffer, loaded on 10 x 10 cm cellulose-coated glass plates, and separated by electrophoresis.
- 1 ⁇ g non- radioactive phosphoserine, phosphothreonine and phosphotyrosine were used as internal standards and vis ualized by ninhydrin staining.
- the radioactive phosphoamino acids were detected by autoradiography. The treatment for each sample is indicated below their loading points.
- Fig. 8 shows reverse transcription-PCR analysis of MAFA expression.
- Total RNA (2 ⁇ g) derived from the indicated rat tissues and from RBL-2H3 cells were reverse transcribed in a reaction mixture containing both (-actin and MAFA- specific primers.
- One third of the cDNA was used as a template in either a (-actin (panel A) or a MAFA (panel B) oligonucleotides-primed PCR reaction.
- One te'nth of each PCR reaction mixture was analyzed by electrophoresis through 1% agarose gels.
- the DNA of the gel shown in panel B was transferred to a nylon membrane, probed with a MAFA-specific internal oligonucleotide in an hybridization solution containing 20% formamide. The membrane was then washed in 0.2X SSC, 0.1% SDS and autoradiographed for 2 hours (panel C).
- the present invention concerns a DNA sequence encoding MAFA, a modulator of the type I Fc ⁇ RI-mediated activation of mast cells and basophils.
- a eukaryotic cDNA expression library was constructed in pcDNA I (Invitrogen) , the cDNA being derived from the poly A+m RNA of the RBL-2H3 cell line, from which there was isolated and subsequently sequenced the cDNA molecule which encodes MAFA (the mafa cDNA) .
- MAFA is a type II integral membrane glycoprotein that is in fact, a new member of the superfamily of C-type animal lectins.
- Naturally-occurring MAFA is a membrane-bound protein and it is not soluble.
- one of the aspects of the present invention is a method to screen for potential ligands which bind specifically to MAFA, and which ligands alone, in a mixture, or in combination with MAFA may be useful to prevent inflammation and allergy.
- screening potential ligands it is, however, necessary to first provide a soluble form of MAFA, preferably one which has retained the binding specificity of MAFA as determined by an assay with monoclonal antibodies to MAFA, e.g. mAb G63.
- obtention of a soluble form of MAFA also enables the generation of additional mAbs specific for MAFA which bind epitopes of MAFA different from those of mAb G63, and which may be useful, for example, for use in the above screening procedure, i.e. they can be used in a competitive binding assay with potential ligands to determine the binding specificity and affinity of such ligands to MAFA.
- Preparation of the cloned cDNA sequence encoding MAFA provides the means for obtaining a soluble form of MAFA.
- the usual standard techniques of recombinant DNA technology may be employed (see, for example, Sambrook et al., 1989) .
- the cloned cDNA encoding the mafa sequence depicted in Fig. 5 can be manipulated by addition, deletion or substitution mutagenesis procedures using standard methods (Sambrook et al., 1989) to generate a series of modified MAFA-encoding sequences and recombinant vectors containing them, which when expressed in a suitable host, will provide a series of MAFA derivatives, differing from one another and from the native MAFA by having more than one amino acid residues added, deleted or substituted with another amino acid residue.
- MAFA derivatives may then be analyzed by standard procedures to ascertain whether or not they are soluble. Should there arise a situation where suitable soluble MAFA derivatives are not obtained, then it is also possible to convert, by known methods, either the native MAFA or non-soluble MAFA derivatives into soluble forms.
- MAFA may therefore also be manipulated to obtain the soluble extracellular domain of MAFA, this being a soluble derivative of MAFA.
- a target sequence that is cleavable by specific proteases e.g., the blood coagulation factor Xa (the tetrapeptide sequence Ile-Glu-Gly-Arg) , (Nagai and Thogersen, 1987) into the extracellular domain of MAFA close to the transmembranal stretch of MAFA.
- a target sequence that is cleavable by specific proteases, e.g., the blood coagulation factor Xa (the tetrapeptide sequence Ile-Glu-Gly-Arg) , (Nagai and Thogersen, 1987) into the extracellular domain of MAFA close to the transmembranal stretch of MAFA.
- Such a recombinant vector encoding the so- modified mafa sequence by the appropriate host cells, transfected with this vector, such as, for example, prokaryotic, insect or mammalian cells, will enable the production of the extracellular domain of MAFA, i.e. a soluble form of MAFA, once these cells are treated with the respective protease, e.g. factor Xa.
- the above transfected cells would express MAFA as a membrane-bound protein and when treated with the protease, the extracellular portion of MAFA will be cleaved at the target sequence site resulting in the release of this extracellular portion of MAFA into the culture medium from which it may be collected and purified.
- a leader sequence may be, for example, the hydrophobic leader sequence of the glycoprotein D (gD-1) from herpes simplex virus type I (HSVI) (Caras et al., 1987) .
- gD-1 glycoprotein D
- HSVI herpes simplex virus type I
- the expression of such a modified MAFA sequence by the appropriate transfected cells yields a fusion protein, being essentially the extracellular domain of MAFA, i.e. a soluble form of MAFA, that is constitutively secreted (for constitutive secretion of such fusion proteins, see Caras et al., 1987) into the culture medium from which it may be collected and purified.
- the above noted manipulation and subsequent expression of the mafa sequence in the host cells are carried out by standard procedures (Sambrook et al., 1989).
- the cDNA encoding native MAFA or any of the above mentioned modified MAFA-encoding sequences can be expressed in eukaryotic or prokaryotic host cells that are transformed by a vector containing the mafa cDNA or modified (deletion or substitution) mafa cDNA.
- Eukaryotic host cells are preferable in view of the fact that MAFA is a homodimeric glycoprotein, and correct post-translational processing, e.g. dimerization and glycosylation take place only in eukaryotic host cells.
- MAFA derivatives some of which may be soluble, in eukaryotic host cells so that these too have the correct post-translational processing.
- prokaryotic expression of MAFA is useful for the production of large amounts of MAFA and MAFA derivatives.
- Suitable eukaryotic host cells may be any of the well known yeast, insect or mammalian cell lines.
- the monkey COS-7 cell line was found to be suitable for experimental MAFA expression in accordance with the present invention.
- suitable cells such as CHO and insect cells, may be used.
- the standard procedures of affinity chro atography may be employed by using a suitable, known solid support or matrix to which is coupled a MAFA-specific monoclonal antibody, e.g. mAb G63.
- the purified soluble derivatives of MAFA may be coupled to a solid support or matrix for use in screening for ligands that are specific to MAFA.
- Potential MAFA-ligands e.g. simple or complex saccharides, proteins, glycoproteins, lipopolysaccharides or glycolipids will be brought into contact with such a MAFA-coupled solid support, and subsequently the ligand(s) which bind to MAFA may be specifically eluted, purified and their chemical na ture analyzed, all by way of standard biochemical procedures. In this way, it will be possible to identify the naturally-occurring MAFA-specific ligands as well as any other MAFA-specific ligands, e.g. synthetically produced sugars.
- a number of methods may be employed, as noted above.
- An example of one procedure for preparing a soluble MAFA derivative and its subsequent use to screen for MAFA-specific ligands is as follows: (i) inserting all or part of the mafa sequence depicted in Fig. 5 in an appropriate commercially available vector able to direct the expression of the MAFA as a soluble protein in the form of a tagged fusion protein;
- MAFA-ligand and eluting the isolated MAFA-ligand for the analysis of its chemical nature by standard biochemical procedures.
- RBL-2H3 cells were obtained from Dr. R._ P. Siraganian, HIH, Bethesda, MD (Barsumian et al. , 1981). They
- SUBSTITUTE SHEET (RULE 26 ⁇ were maintained in Eagle's minimal essential medium with Earle's salts (MEM, GIBCO, Grand Island, NY, USA) supplemented with 10% FCS (GIBCO) , 2 mM glutamine, and antibiotics (penicillin-streptomycin mixture, Bio-Lab, Jerusalem, Israel) .
- the G63 hybridoma cells (Ortega and Pecht, 1988) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 2 mM sodium pyruvate (Bio-Lab) , and antibiotics.
- KU812 cells (kindly provided by Dr. E. Razin, Hebrew University of Jerusalem) and COS-7 cells were maintained in DMEM, supplemented with 10% FCS and antibiotics.
- P815 cells (obtained from Dr. R. Levi, The
- mAb G63 (IgGl) was purified from G63 hybridoma culture supernatants by chromatography on protein A- Sepharose (Pharmacia, Uppsala, Sweden) . The mAb was eluted with 0.2 M sodium citrate (pH 4.5) directly into tubes containing neutralizing 2 M Tris buffer (pH 8.2) . The mAb was dialyzed against 0.1 M HEPES pH 8.0, and stored at - 20°C.
- the chloramine-T method was used for labeling with [1251] -sodium iodide (Amersham, UK) .
- the specific activity obtained was typically 6700 cpm/fmol.
- RBL-2H3 and COS-7 cells were surface iodinated by the lactoperoxidase technique (Marchalonis, 1966) .
- PBS lactoperoxidase
- the cells were solubilized by 0.5% Triton X-100 in 10 mM HEPES, 10% glycerol, 0.15 M NaCl (pH 7.6) in the presence of 2mM phenyl ethyl-sulfonyl fluoride, 1 ⁇ g/ l pepstatin, 2 -(g/ml leupeptin, and 10 mM iodoacetamide in a final volume of 1.5 ml. After 15 min on ice, non-solubilized material was removed by centrifugation at 20,000 x g for 10 min at 4°C. (e) Immunoprecipitation and deglycosylation.
- mAb G63 was coupled to Affigel-10 (Bio-Rad, Richmond, CA, USA) , as recommended by the manufacturer, at a ratio of 10 mg of antibody per ml of Affigel-10.
- 10-50 ⁇ l of mAb G63-coated beads were added to the cell lysates and the suspensions were gently rocked at 4(C for 4 hours. The beads were then washed three times batchwise with a buffer containing 0.05% Triton X-100 in 10 mM HEPES, 10% glycerol and 0.5 M NaCl (pH 7.6) .
- the beads were either resuspended in deglycosylation buffer (see below) or boiled in SDS-PAGE sample buffer (9% SDS, 30% glycerol, 0.02% brom ⁇ phenol blue, 185 mM Tris-Cl pH 6.8) for elution and electrophoresis of the bound MAFA.
- SDS-PAGE sample buffer 10% SDS, 30% glycerol, 0.02% brom ⁇ phenol blue, 185 mM Tris-Cl pH 6.8
- the G63-coated beads carrying the immunoadsorbed MAFA were resuspended in 50 ⁇ l Tris-Cl 10 mM pH 7.0 containing 0.1% SDS and 0.5% ⁇ - mercaptoethanol, and boiled for 5 min.
- G63 immunoprecipitates of surface-labeled RBL-2H3 cell lysates were deglycosylated and submitted to reducing SDS-PAGE, after which wet electrophoretic transfer to nitrocellulose was performed in a buffer containing 25 mM Tris, 190 mM glycine and 10% methanol. The bands of interest were then visualized by autoradiography and the corresponding nitrocellulose strips were excised and incubated with 0.5 units of endoproteinase Lys-C at 37°C for 24 hours in a volume of 50 ⁇ l.
- Homogenates derived from 109 RBL-2H3 cells by the proteinase K/SDS method (Gonda et al., 1982) were stirred for 2 hours at room temperature with approximately 0.1 of oligo dT- cellulose (Boehringer Mannheim) preswollen in high salt buffer containing 0.5 M NaCl, 0.1% SDS, 1 mM EDTA and 10 mM Tris-Cl pH 7.5.
- the oligo dT-cellulose was collected by centrifugation and washed twice with high salt buffer
- SUSSfITUTESHEET(RULE26 ) containing LiCl instead of NaCl, and twice again with the high salt buffer described above.
- the oligo dT-cellulose was poured in a sterile disposable column and the poly A+ mRNA was eluted stepwise with low salt buffer containing 0.05% SDS, 1 mM EDTA and 10 mM Tris-Cl pH 7. 5.3 ⁇ g of poly A+ mRNA were converted to double-stranded cDN as described (Gubler and Hoffman, 1983) .
- COS-7 cells grown on Flaskette glass slide chambers were separately transfected with 3 (g of each plasmid pool using the DEAE-dextran / chloroquine method essentially as described elsewhere (Seed and Aruffo, 1987) .
- the ligand binding assay on the transfected monolayers was performed by replacing the growth medium with 1.5 ml fresh medium containing the radioiodinated mAb G63 (6700 cpm/fmol, InM) .
- the Flaskettes were then placed on an orbital shaker at slow motion for 2 hours at room temperature after which the cells were extensively washed by 6 changes of ice-cold PBS.
- DNA sequencing was performed using the Taq Polymerase-Dye Deoxynucleotide reaction kit (Applied Biosystems) and an Applied Biosystems 373A sequencer and software. Overlapping sequences from both strands were determined and the compilation of the sequence information from individual reactions was done using the Inherit application program and SeqEd sequence editor (Applied Biosystems) . Sequence analysis was performed using the University of Wisconsin Genetics Computer Group software package (Version 7.2) .
- cDNA synthesis and PCR amplification were performed basically as described (Pinkas- Kramarski et al., 1994) .
- the MAFA primers used were oligonucleotides 8077 (5' GCCACTGTTACTACTTCT 3') and 8075 (5' GACCTTCTCACAGATCCA 3') .
- the primers for rat ⁇ -actin were as described (Jin et al., 1993).
- the first strand of cDNA was synthesized at 37°C in a reaction mixture that contained reverse transcriptase (5 units, Promega) , both MAFA and (-actin antisense oligonucleotides (10 pmol each) and RNA (2 -]g) .
- reverse transcriptase 5 units, Promega
- MAFA reverse transcriptase
- MAFA monomerase-dependent adenosine-dependent RNA sequence
- RNA (2 -]g RNA (2 -]g
- One quarter of the reaction mixture was used as template for each of two parallel PCR reactions; one of them containing 10 pmol of both (-actin primers, and the other 10 pmol of both MAFA primers.
- the cycling conditions were set as described (Pinkas-Kramarski et al., 1994) .
- the products (one tenth of the reaction mixture) were resolved by electrophoresis through a 1.2% agarose gel.
- the MAFA PCR amplification products were capillary transferred to a nylon membrane and probed using an end- labeled internal oligonucleotide (#7596; 5' GGAGTATGTGGGCGAGG 3') at 42°C in the presence of 20% formamide.
- the blot was washed at 42 (C with 0.2 x standard saline citrate (SSC) and 0.2% sodium dodecyl sulfate, and autoradiographed.
- SSC standard saline citrate
- sodium dodecyl sulfate sodium dodecyl sulfate
- Samples scheduled for antigenic stimulation were primed by the addition of 10 nM DNP-specific, IgE class mAb (SPE-49) simultaneously with the orthophosphate, and stimulated after the 3 hours long phosphate-incorporation period by the addition of 100 ng/ml DNPll-BSA for two minutes.
- 10 nM mAb G63 was added to the cells 10 minutes prior to lysis or antigen stimulation.
- the above treatments were stopped by two fast washings with ice-cold PBS and the immediate addition of 1ml of the above described lysis buffer supplemented with 100 mM NaF, 2 mM sodium orthovanadate and 10 mM sodium pyrophosphate.
- the cell lysates were scraped with a rubber policeman and transferred to 1.5 ml test tubes.
- Cell debris were sedimented by centrifugation (1 minute at 15000 rpm in a Beckman microfuge) and the clear supernatants were incubated with a combination of 20 ⁇ l of mAb G63-coated agarose beads and 4 ⁇ l of protein A-Affigel (Bio Rad) for 3 hours at 4(C with gentle rocking. After sedimenting, the immunobeads samples were washed as described above and further incubated with N-glycosidase F for 16 hours.
- PVDF polyvinylidene difluoride
- the hydrolysates were then dissolved in 5 ⁇ l pH 3.5 buffer (glacial acetic acid:pyridine:water, 5:0.5:94.5, v/v/v) containing 1 ⁇ g phosphoserine, phosphothreonine and phosphotyrosine.
- the phosphoamino acids were separated by electrophoresis on cellulose coated glass plates (Merck) in pH 3.5 buffer at 1 kV for 25 minutes. The plates were then air-dried and the phosphoamino acid standards were visualized upon spraying with 0.2% (w/v) ninhydrin in ethanol.
- the radioactive phosphoamino acids were then detected by autoradio-graphy and quantified by densitometric analysis.
- Example 1 The Mr of the MAFA polypeptide core is 20 kDa.
- N-linked oligosaccharide side chains of the MAFA In order to assess the content of N-linked oligosaccharide side chains of the MAFA, a mAb G63-coated beads-immunoprecipitate obtained as before from surface- iodinated cells was incubated in the presence of N- glycosidase F to remove asparagine-linked oligosaccharide side chains, eluted by boiling in SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Under non-reducing conditions, two considerably narrow bands, of Mr 18 and 40 kDa, respectively, were resolved (Fig. 1 lane 1) . Hence, the N- linked oligosaccharide side chains account for up to half of the apparent molecular mass of the MAFA.
- Example 2 Subunit composition of the MAFA.
- peptide maps were prepared from tryptic proteolysis fragments derived from both N-deglycosylated 18 and 40 kDa forms of the MAFA. These were first visualized by autoradiography and the correspondent radioactive bands were excised from the polyacrylamide gel. The protein contained in these gel pieces was then further radioiodinated with the chloramine T method.
- a cDNA eucaryotic expression library was constructed using polyadenylylated mRNA isolated from RBL- 2H3 cells, which consti utively express the MAFA glycoprotein.
- 65 pools of 10,000 cDNA clones each were transfected into COS-7 cells on glass-bottom chambers and the monolayers were screened for 1251-labeled mAb G63- binding cells, performing emulsion autoradiography followed by darkfield microscopy, as described in Experimental Procedures.
- One of the 65 cDNA pools conferred on the transfected COS-7 cells the ability to bind the radioiodinated mAb G63.
- the binding specificity was established by its complete inhibition in the presence of a 100 fold excess of unlabeled mAb. Further, it was also unaffected by the addition of a similar excess of an isotype-matched IgGl antibody (J17; (Ortega et al., 1988)). Subpooling of AO, and of the positive pools derived thereof, led to the isolation of a positive clone.
- the expression of the cloned cDNA by transfected COS-7 cells was analyzed by flow cytometry following sequential incubation with biotinylated mAb G63 and phycoerythrin- labeled streptavidin. 38% of the COS-7 cells used for transfection were stained with mAb G63 (Fig.
- mAb G63 immunoprecipitated from the transfected cells' lysates a single labeled species with an apparent Mr of 58 kDa under non-reducing conditions and 28 kDa under reducing conditions.
- Mr of the respective proteins isolated from the RBL-2H3 and transfected cells might be due to differences in post- translational modifications. Therefore, the apparent molecular mass of the respective N-deglycosylated polypeptide chains were compared. Lysates from surface- radioiodinated RBL-2H3 cells and COS-7 cells transfected with the cloned cDNA were incubated with mAb G63-coated beads.
- the immunoprecipitates were N-deglycosylated, eluted by boiling in SDS-PAGE sample buffer and analyzed by electrophoresis under reducing conditions. Both protein samples were shown by autoradiography (Fig. 4A) to have a 20 kDa polypeptide core, further suggesting that the cloned protein is, indeed, MAFA. To substantiate this point, both 20 kDa polypeptides shown in Fig. 4A were digested in parallel with endoproteinase Lys-C. Analysis of the resulting peptide fragments by tricine-SDS gel electrophoresis and autoradiography revealed in both cases a single radioactively labeled peptide of 2.6 kDa (Fig. 4 B) . Taken together, these results strongly support that the cloned cDNA sequence indeed codes for the MAFA.
- nucleotide sequence of the MAFA cDNA is depicted in Fig 5.
- An open reading frame deduced from the nucleotide sequence starts at nucleotide # 54 with a codon for methionine and ends at nucleotide # 617 before a TGA stop codon.
- the other C-type lectins displaying high sequence homology with the MAFA CRD are all involved in immunological functions. These are the type II receptor for IgE (Fc ⁇ Rli / CD23) (Bettler et al., 1989), the natural killer antigen NKR-P1 (Giorda and Trucco, 1991) , the T-cell early activation antigen CD69 (Hamann et al., 1993), the B-cell differentiation antigen CD72 (von Hoegen et al., 1990), and Ly-49, an NK cell receptor to MHC class I alloantigens (Chan and Takei, 1989) .
- serine 8 is part of a tyr-ser-thr-leu sequence, and related Y-x-x-L/I motifs were observed in the cytoplasmic domain of several type II integral membrane proteins of the C-type animal lectin family (Table 1) .
- the leucine residue located (in most cases) three amino acids carboxyl-terminal to each of the tyrosines also appears to be essential for that motifJEs function (Samelson and Klausner, 1992) .
- Phosphoamino acid analysis of the MAFA was undertaken in samples derived from resting and Fc ⁇ RI stimulated cells, both in the presence and the absence of MAFA clustering by mAb G63.
- RBL-2H3 cells cultured in medium containing 32P-labeled orthophosphate, were IgE saturated by the presence of 10 nM of a DNP-specific mouse monoclonal antibody and stimulated for 2 minutes by the addition of 100 ⁇ g/ml DNPll-BSA.
- the effect of the MAFA clustering was examined by the addition of 10 nM mAb G63 to the cells 10 minutes prior to lysis (non-stimulated cells) or to the addition of antigen (activated cells) .
- G63- immunoprecipitates from the lysates were first incubated with N-glycosidase F and then eluted and separated through a 15% SDS-polyacrylamide gel under reducing conditions. The proteins were then electrotransferred to a PVDF membrane and visualized by autoradiography (not shown) . The 20 kDa N- deglycosylated MAFA contained in the PVDF strips was then hydrolyzed by heating to 110°C in 6 N HCl for 1 hour. The phosphoamino acids were separated by electrophoresis on cellulose-coated plates, which were then autoradiographed (Fig. 7) .
- control phosphoserine phosphothreonine and phosphotyrosine was determined by ninhydrin staining. No 32P labeled phosphothreonine could be observed in any of the samples. In contrast, similar amounts of radioactive phosphoserine and phosphotyrosine were found in samples derived from resting cells, indicating that the MAFA is constitutively phosphorylated on both seryl and tyrosyl residues. Antigen stimulation of RBL-2H3 cells, as well as their incubation in the presence of mAb G63, slightly increased the intensity of phosphorylation of both these residues.
- Example 6 Transcription of the MAFA gene in different rat organs.
- Reverse transcription-polymerase chain reaction is a combined method which allows a very sensitive detection of the presence of a given sequence in a relatively small sample of RNA.
- 2 ⁇ g of total RNA derived from rat liver, kidney, muscle, lung, brain, thymus, spleen, PeyerJBs patches, blood cells and from RBL-2H3 cells were reverse transcribed in reactions primed by both MAFA and -actin- derived primers (Pinkas-Kramarski et al., 1994).
- One third of the produced single standard cDNA was used as a template for each of two PCR reactions designed to amplify ⁇ -actin and MAFA-derived sequences, respectively.
- the DNA was then transferred to a nylon membrane, which was probed with an internal MAFA-derived oligonucleotide.
- the probe hybridized to the 308 bp long amplification products.
- the presence of MAFA-derived PCR products was also detectable in the other samples as well (not shown) , indicating that although MAFA transcripts are readily detectable in the mentioned rat organs, very small amounts appear to be present also in the other examined organs (i.e. kidney, muscle, brain, thymus, peyer patches and blood cells) .
- Brain neurons and glial cells express Neu differentiation factor / heregulin: a survival factor for astrocytes. Proc. Natl. Acad. Sci. USA, 91, 9387-9391.
- a murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J. Immunol., 143, 1379-1386.
- Lys Gly Ser Lys Gin Trp lie Gin Ala Arg Phe Ala Cys Ser Asp Leu 20 25 30
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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EP95916215A EP0759940A4 (en) | 1994-04-08 | 1995-04-06 | A dna molecule encoding a mast cell function-associated antigen (mafa) |
US08/722,126 US6034227A (en) | 1995-04-06 | 1995-04-06 | DNA molecule encoding a mast cell function-associated antigen (MAFA) |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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IL10925794A IL109257A (en) | 1994-04-08 | 1994-04-08 | Dna molecule encoding a mast cell function-associated antigen (mafa) |
IL109257 | 1994-04-08 |
Publications (1)
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WO1995027734A1 true WO1995027734A1 (en) | 1995-10-19 |
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PCT/US1995/004258 WO1995027734A1 (en) | 1994-04-08 | 1995-04-06 | A dna molecule encoding a mast cell function-associated antigen (mafa) |
Country Status (3)
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EP (1) | EP0759940A4 (en) |
IL (1) | IL109257A (en) |
WO (1) | WO1995027734A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998004585A2 (en) * | 1996-07-31 | 1998-02-05 | Incyte Pharmaceuticals, Inc. | Novel human macrophage antigen |
WO1998054209A2 (en) * | 1997-05-31 | 1998-12-03 | Peptide Therapeutics Limited | Human mast cell function-associated antigen (mafa) and uses thereof |
WO2001070805A2 (en) * | 2000-03-17 | 2001-09-27 | Gemini Science, Inc. | Soluble mast cell function associated antigen (mafa) pharmaceutical compositions and methods of making and using them |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US4683135A (en) * | 1983-07-27 | 1987-07-28 | Yeda Research And Development Co., Ltd. | DSCG binding protein and process for preparing same |
-
1994
- 1994-04-08 IL IL10925794A patent/IL109257A/en active IP Right Grant
-
1995
- 1995-04-06 EP EP95916215A patent/EP0759940A4/en not_active Withdrawn
- 1995-04-06 WO PCT/US1995/004258 patent/WO1995027734A1/en not_active Application Discontinuation
Non-Patent Citations (3)
Title |
---|
INTERNATIONAL IMMUNOLOGY, Volume 3, Number 4, issued 1991, ORTEGA et al., "Possible Interactions Between the Fc(epsilon) Receptor and a Novel Mast Cell Function-Associated Antigen", pages 333-342. * |
JOURNAL OF IMMUNOLOGY, Volume 141, Number 12, issued 15 December 1988, ORTEGA SOTO et al., "A Monoclonal Antibody that Inhibits Secretion from Rat Basophilic Leukemia Cells and Binds to a Novel Membrane Component", pages 4324-4332. * |
See also references of EP0759940A4 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998004585A2 (en) * | 1996-07-31 | 1998-02-05 | Incyte Pharmaceuticals, Inc. | Novel human macrophage antigen |
WO1998004585A3 (en) * | 1996-07-31 | 1998-03-12 | Incyte Pharmaceutical Inc | Novel human macrophage antigen |
US6034219A (en) * | 1996-07-31 | 2000-03-07 | Incyte Pharmaceuticals, Inc. | Human macrophage antigen |
WO1998054209A2 (en) * | 1997-05-31 | 1998-12-03 | Peptide Therapeutics Limited | Human mast cell function-associated antigen (mafa) and uses thereof |
WO1998054209A3 (en) * | 1997-05-31 | 1999-03-11 | Peptide Therapeutics Ltd | Human mast cell function-associated antigen (mafa) and uses thereof |
WO2001070805A2 (en) * | 2000-03-17 | 2001-09-27 | Gemini Science, Inc. | Soluble mast cell function associated antigen (mafa) pharmaceutical compositions and methods of making and using them |
WO2001070805A3 (en) * | 2000-03-17 | 2002-03-14 | Gemini Science Inc | Soluble mast cell function associated antigen (mafa) pharmaceutical compositions and methods of making and using them |
US7056677B2 (en) | 2000-03-17 | 2006-06-06 | Gemini Science, Inc. | Soluble mast cell function associated antigen (MAFA) pharmaceutical compositions and methods of making and using them |
Also Published As
Publication number | Publication date |
---|---|
EP0759940A1 (en) | 1997-03-05 |
IL109257A0 (en) | 1994-07-31 |
IL109257A (en) | 2005-03-20 |
EP0759940A4 (en) | 1999-04-14 |
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