Classifications

• • 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
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)

Definitions

• This invention is in the field of recombinant f 5 DNA technology and more particularly to the production of
• IgE specific protein having an apparent molecular weight of about 31,000 daltons.
• IgE immunoglobulin E
• IgE-binding proteins include the cell surface receptors of mast cells, basophils, lymphocytes and other cell types (1,2). The receptors on mast cells and basophils are responsible for
• IgE-mediated immediate hypersensitivity reactions (3,4), while those on lymphocytes play important roles in the regulation of IgE antibody responses (5,6).
• Another type of IgE-binding proteins includes the lymphokines which function in either potentiating or suppressing IgE
• this invention's primary objective is to initiate molecular cloning of DNA for these relevant proteins.
• Rat basophilic leukemia are cells which have been used extensively to study a high affinity IgE
• the primary object of the present invention is to create and culture recombinant DNA molecules which produce an IgE specific protein having an approximate molecule weight of about 31,000 daltons.
• the long-range objective is to use this in vitro derived protein to establish the structural relatedness of the IgE system proteins, the structure-function relationship of each of these proteins, and the regulation of their gene expression.
• Recombinant DNA molecules are produced which have the translatable end-product of an apparently 31,000 dalton IgE specific protein.
• double-stranded cDNA was synthesized from sucrose gradient-fractionated RBL mRNA, inserted into vector plasmids and used to transform recipient host organisms.
• By screening transformants using a hybridization-selection /in vitro translation procedure several clones containing cDNA which hybridized to mRNA coding for a 31,000 IgE-binding protein were identified.
• the DNA sequence of one of these cloned cDNAs was determined and the amino acid sequence corresponding to the part of the protein was deduced.
• This cloned cDNA most likely codes for the 31,000 IgE-binding protein identified in RBL, cells, which appears to be uniquely related to the IgE-binding phenotype of the cells and which may have a significant role in the IgE mediated activation of basophils and mast cells.
• the invention includes a method of producing a cDNA molecule which has, as a translational product, a polypeptide which selectively binds IgE, the method comprising of isolating the genetic material ⁇ -'coding for IgE-binding polypeptide which has a molecular weight of about 31,000 daltons, is immunoprecipitated from translational products of RBL mRNA, and . is not precipitated by normal rabbit serum; incorporation of the genetic material into a vector; transfer of the vector into a recipient organism; selection and cloning of the host cell carrying the IgE-binding protein expression; producing clones; and collecting the protein produced.
• An IgE-binding protein produced by the aforesaid method reacts specifically with the anti- ⁇ BP antiserum and binds with IgE-Sepharose 4B.
• the IgE-binding protein produced by the aforesaid method is characterized as a molecule with at least 5 methionine residues; a molecule which is devoid of potential N-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE- binding. protein.
• the invention also includes cDNA characterized by the production of an approximately 31,000 Dalton IgE-specific binding protein.
• the cDNA is characterized by the production of a translational product, wherein said translational product is: an approximately 31,000 Dalton IgE-binding protein; a molecule with at least five methionine residues; a molecule having no N-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE protein.
• the cDNA may also be characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, characterized in that it is: a molecule having 570 base pairs; a molecule that reacts specifically with antiepsilon BP antiseru ; and a molecule . which hybridizes specifically with mRNA from RBL cells.
• IgE binding protein having an approximate molecular weight of about 31,000 daltons characterized in that said cDNA's carboxy-terminal half amino acid-coding sequence is substantially:
• the cDNA molecule is characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, said cDNA also being characterized in that it is a molecule with 121 nucleotides in a 3' untranslated region; a molecule with a typical AATAAA sequence 15-21 bases before the poly (A) addition site; a molecule with a sequence devoid of any transmembrane sequences; a molecule with a sequence devoid of any N-linked carbohydrate attachment sites; and a molecule which displays hydrophilic characteristics.
• the cDNA molecule is also characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons wherein said molecule reacts specifically with an RNA species from • RBL cells.
• Figure 1 is a representation of NaDodSo.-PAGE analysis of IgE-binding proteins isolated from the in vitro translation products of RBL mRNA.
• B) In vitro translation products from sucrose gradient-fractionated RNA (pool of fractions 13-16) were subjected to immunoprecipitation with rabbit anti- ⁇ BP serum (lane a) or normal rabbit serum (lane b). The proteins were analyzed on 10% polyacrylamide gels.
• Figure 2 is a representation of NaDodSO.-PAGE analysis of the in vitro translation product from mRNA hybridized to a cloned cDNA.
• mRNA hybridizing with 136C9.13 cDNA immobilized on nitrocellulose filters was translated in vitro and the translation products were reacted with Sepharose 4B conjugated with BSA (lane a), mouse monoclonal IgG, (lane b) or mouse monoclonal IgE. (lane c), or were subjected to immunoprecipitation with rabbit anti- ⁇ BP serum (lane d) , or normal rabbit serum (lane e) .
• Isolated proteins were analyzed on 10% polyacrylamide gels.
• Figure 3 is a representation of the nucleotide sequence of cloned cDNA 136C9.13. The amino acids predicted from the nucleotide sequence are presented below the coding sequence.
• Figure 4 is a reproduction of Northern blot hybridization analysis of RNA from various cell lines with cloned 136C9.13 cDNA probe.
• Total poly(A) + RNA (5 ⁇ g each) from 5 cell lines - a mouse IgE-secreting hybridoma (26.82, lane a), a mouse IgG,-secreting hybridoma (109.3, lane b) , RBL (lane c), mouse mastocytoma P815 (lane d) and rat lymphoma IR983F (lane e) were glyoxylated, electrophoresed on 2% agarose gels, transferred to nitrocellulose filters and hybridized with 32P-labeled
• Figure 5 is a representation of NaDodSO.-PAGE analysis of 125I-labeled IgE-binding protein from RBL cells.
• A Cell lysates from RBL (lane a), P815 (lane b) or rat lymphoma (lane c) were subjected to repetitive affinity purification with IgE-Sepharose 4B as described in Example 1. The proteins were labeled with 125I at the end of the first cycle of affinity purification.
• B RBL cell lysates were subjected to affinity purification with
• IgE-Sepharose 4B and the bound proteins were iodinated, eluted and treated with lentil lectin-Sepharose 4B.
• the unbound material was then i) reacted with IgE-Sepharose 4B (lane a); or ii) subjected to immunoprecipitation with rabbit anti- ⁇ BP '(lane b) or normal rabbit serum (lane c).
• Isolated proteins were analyzed on 10% polyacrylamide gels Detailed Description of the Invention
• This invention involves the production of a IgE binding protein, and the production of a cDNA molecule capable of use in recombinant DNA technology. More particularly, this invention involves the formation of a cDNA that produces a IgE binding protein having an approximate molecular weight of about 31,000 daltons, and the production of host/recipient organisms which are capable of producing that protein.
• Sucrose gradient- fractionated RBL mRNA was subjected to in vitro translation in the rabbit reticulocyte lysate system.
• the translation products (composed of numerous translated proteins — data not shown) were examined by affinity purification with IgE immunoadsorbent. As shown in Figure 1A, a 31K protein was isolated virtually free of other proteins from the translation products of 13-14S RNA.
• a 31K protein was immunoprecipitated from the translation products of RBL mRNA by anti- ⁇ BP serum (lane a) and not by normal rabbit serum (lane b) . It should be noted that the 31K protein band is clearly the major protein in immunoprecipitates and is the only protein which was immunoprecipitated specifically.
• RBL mRNA from sucrose density gradient fractions 14-16 was used to generate a cDNA library which was screened by a hybridization-selection/in vitro translation procedure for clones coding for the IgE-binding protein. From about 1000 clones, one positive clone (136C9.13) was identified. As shown in Figure 2, cDNA from this clone hybridized to mRNA from RBL cells which could be translated to a 31K protein which binds IgE (lane c) but not BSA (lane a) or IgG, (lane b) . Furthermore, the translated protein was shown to react specifically with the anti- ⁇ BP antiserum (lane d vs. lane e).
• the nucleotide sequence of the 136C9.13 cDNA was determined and a single open reading frame was identified. As shown in Figure 3, this cDNA contains a coding sequence for 138 amino acids representing approximately the carboxy-ter inal half of the protein.
• the cDNA sequence also contains 121 nucleotides of 3' untranslated region and a typical AATAAA sequence 15-21 bases before the poly(A) addition site. Homology searches of the nucleic acid database demonstrated that this sequence has not been previously described and the there is no significant homology to other known protein or DNA sequences. The protein appears rather hydrophilic as indicated by a hydropathicity plot (29); inspection of the sequence does not identify any potential transmembrane sequences nor any N-linked carbohydrate attachment sites.
• the 31K IgE-Binding is Expressed Uniquely by RBL Cells and Not by Cells Lacking Receptors.
• the protein bound to and subsequently eluted from the first IgE-Sepharose 4B described above was allowed to react with lentil lectin-Sepharose 4B and the unbound material was then treated with IgE immunoadsorbent'.
• the 31K protein was indeed the major protein isolated by IgE-Sepharose 4B. This result indicates that the 31K protein found in RBL cells is an IgE-binding protein.
• the example of the invention is divided into the following stages:
• IgE HI-DNP- ⁇ -26.82
• IgG- j ⁇ HI-DNP- ⁇ l-109.3 specific for DNP
• GAG goat anti-rabbit IgG
• rabbit antiserum prepared against proteins affinity- purified from RBL cells by repetitive chromatography on
• IgE immunoadsorbent (anti- ⁇ BP; 15) have been described previously. Immunoadsorbents were prepared by conjugating
• RNA and In Vitro Translation of RNA Extraction of total cytoplasmic RNA from various cell sources by the standard phenol/chloroform method, isolation of poly(A) RNA by oligo(dT)-cellulose affinity chromatography and sucrose density gradient fractionation of RBL poly(A) RNA have all been described (22). In vitro translation of mRNA was performed using the rabbit reticulocyte lysate system (New England Nuclear) in the presence of [ 35S]methionine.
• cDNA Cloning and Screening Double-stranded cDNA was prepared from sucrose gradient-fractionated RBL mRNA, inserted into the Pst I site of pBR322 and used to transform E. coli C600 (23).
• a hybridization-selection/in vitro translation procedure was used to screen for positive clones (24). Briefly, pools of 12 chimeric plasmids were bound to nitrocellulose filters, the filters were incubated with total RBL poly (A) RNA and the hybridized RNA was eluted and translated in vitro. The translation products were subjected to immunoprecipitation (see below) to detect an IgE-binding protein. A single positive clone was identified by subjecting the individual clones of a positive pool to the same procedure. This cell line, identified as 136C9.13, is on deposit in the American Type Culture Collection, accession No. and will be available under the terms of the Budapest Treaty.
• the beads were washed four times with 4 ml of TENT-1% Triton X-100, and one time with 4 ml of TENT-0.05% NaDodS0 4 .
• the bound protein was eluted with 75 ⁇ l of 62.5 mM Tris-HCl, pH
• the cell lysate (typically 5 x 10 cells) was subjected to the first cycle of affinity purification as described in (A) , and the bound proteins were eluted three times each with 200 ⁇ l of 0.5N acetic acid/1% Triton X-100, neutralized with 75 ⁇ l of 2 M Tris-HCl, pH 8.8/1 Triton X-100 and subjected to a second cycle of affinity purification.
• proteins were radiolabeled while still bound to IgE-Sepharose 4B by the chloramine-T method (25) using 1 mCi of Na 125I and 10 ⁇ g of chloramine-T in 30 ⁇ l of 0.1 M sodium phosphate, pH 7.5, for 60 seconds at 4°C.
• the beads wre further washed three times with 4 ml of TENT-1% Triton X-100 and the bound proteins were eluted as described above.
• RNAs were glyoxylated, electrophoresed on 2 % agarose gels, transferred to nitrocellulose filters and hybridized with cDNA probe labeled with 32p by nick-translation (27) hereby incorporated by reference.
• the DNA sequence was determined by the dideoxy primer-extension method._(28) hereby incorporated by reference.
• a 31K IgE-binding protein can be translated in vitro from RBL mRNA and has been identified; (2) cDNA coding for this protein has been found to be novel; and (3) this protein has been shown to be expressed specifically in certain cell types known to possess IgE-specific receptors.
• Studies using the cDNA probe as well as antibodies against synthetic peptides corresponding to specific parts of the predicted sequence should further establish the expression of the described gene specifically in IgE-receptor positive cells, the localization of the protein in these cells and the role this protein may play in IgE-mediated activation of basophils and mast cells.
• the protein may be used as an IgE regulant or to absorb IgE, in effect an "IgE sponge", in the handling of allergenic reactions.
• the genetic material used to form the cDNA maybe induced by inserting other human cell lines containing the IgE-specific binding receptors or with other cell material other than RBL mRNA. While the invention has been described in terms of rat IgE-binding protein, the methods are equally applicable to producing cDNA's useful in detecting related human equivalents or siderophones.

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