Abstract:
The invention relates to the allergenic latex protein Hev b 4. The full cDNA sequence of the smaller protein components of Hev b 4 is disclosed, together with the amino acid sequence of those components and the synthesis of a recombinant protein using the aforesaid DNA sequence or a synthetic peptide derived from the amino acid sequence. The use of the Hev b 4 protein complex and of an antibody against that protein is described in immunoassays and in immunotherapy for the relief of allergy or allergy symptoms.

Description:
[0001]     This invention relates to the allergenic latex protein Hev b 4. More particularly it relates to the full cDNA sequence of the smaller protein components of Hev b 4, the amino acid sequence of those components and the use of the Hev b 4 protein complex in immunoassays and in immunotherapy.  
       BACKGROUND OF THE INVENTION  
       [0002]     Proteins constitute about 1-2% fresh weight of  Hevea brasiliensis  latex. About 70% of latex proteins are found in the aqueous phase, with the remainder being associated or bound to organelles including rubber particles. A small proportion of these latex proteins are known to be allergenic in that they can induce an allergic response in sensitised persons (Yeang et al, 2002). At present, there are thirteen allergenic latex proteins recognised by the International Union of Immunological Societies (IUIS). These are named Hev b 1 to Hev b 13 according to the IUIS-World Health Organisation nomenclature for allergens.  
         [0003]     In the original patent application on latex allergens filed in Malaysia (PI 9402468, 16 Sep. 1994) and subsequently in other countries (e.g. EP-A-0704457), three latex allergens were claimed. These were Hev b II, Hev b III and Hev b IV, which, under the revised IUIS nomenclature, are presently known as Hev b 2, Hev b 3 and Hev b 4 respectively. In the patent specification, Hev b 4 was described as being a component of the latex microhelix complex located in a latex organelle known as the lutoid. Hence, Hev b 4 is a protein complex, rather than a single protein. When the protein complex is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), it separates into three components, with the largest protein discerned at 56 kDa followed by two smaller proteins of about 50 kDa. The allergenicity of all three components of the protein complex was demonstrated by their ability to bind IgE antibody of latex allergic patients&#39; sera (Sunderasan et al., 1995). The N-terminal sequence of one of the Hev b 4 components appeared in the aforementioned Malaysian patent specification, but the sequence did not match any of the known proteins in the databases at the time. The complete amino acid sequence of any of the three proteins constituting Hev b 4, and the cDNA sequences encoding these proteins were not available at the time the patent was filed. In subsequent research, it was shown that the larger protein component was a cyanogenic β-glucosidase (Sunderasan et al., 2002). The smaller protein components (which later research would show to be the main allergenic components of Hev b 4) remained unelucidated.  
       SUMMARY OF THE INVENTION  
       [0004]     In the current patent application, the full cDNA sequence of the smaller protein components (approx. 50 kDa) of Hev b 4 is presented. From the DNA sequence, the complete amino acid sequence of the smaller protein components of Hev b 4 is deduced. Evidence is presented to show that although both the larger and smaller components of Hev b 4 are allergenic, prevalence of sensitisation to the smaller components is higher as compared with the larger component (β-glucosidase). 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0005]      FIG. 1  shows the DNA sequence (1375 bp) of the full-length cDNA clone encoding the smaller components of the  Hevea brasiliensis  latex allergenic protein complex, Hev b 4, where a, g, c and t are the abbreviations for the nucleotide bases adenine, guanine, cytosine and thymine respectively. The potential translation start codon (ATG) and the stop codon (TAA) of the predicted open reading frame are marked in bold.  
         [0006]      FIG. 2  shows the amino acid sequence of the smaller components of Hev b 4 derived from the translation of the corresponding cDNA as shown in  FIG. 1 . The letters of the alphabet are accepted codes for the amino acids (Cohn, 1984).  
         [0007]      FIG. 3  shows Hev b 4 protein complex and its Western blot after separation by SDS-polyacrylamide gel electrophoresis. The protein is stained with Coomassie Blue to show the presence and electrophoretic migration of Hev b 4 (lane 1). Normal human sera showing no IgE binding to Hev b 4 blot (lane 2) serves as a negative control. Binding of human IgE of patients sensitised to Hev b 4 on the Western blot (lanes 3, 5, 7, 9, 11, 13, 15 and 17). Only two patients (lanes 3 and 15) from the eight positive to Hev b 4 show strong IgE binding to the larger component of Hev b 4. Latex allergic patients&#39; sera not reacting to Hev b 4 (Lanes 4, 6, 8, 10, 12, 14, 16, 18, 19 and 20). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0008]     The present invention relates to a cDNA sequence of the smaller components of Hev b 4, a protein complex isolated from the B-serum of centrifuged latex obtained by tapping the rubber tree, H brasiliensis. The following description also details how the N-terminal protein sequence information of the smaller components can be used in cloning the corresponding cDNA, and how the protein can be used in immunoassay and immunotherapy.  
         [0000]     Amino Acid Sequencing of the Protein  
         [0009]     The two protein bands comprising the smaller components of Hev b 4 were excised following SDS-PAGE. N-terminal sequencing performed on the first protein band of the smaller components of Hev b 4 yielded a stretch of forty amino acid residues (shown below); an extension of twenty residues compared to the sequence submitted in the original patent application were obtained. N-terminal sequencing of the second band of the smaller components of Hev b 4 was terminated after the seventh residue, as they appeared identical to that of the first band. The single alphabet representations are accepted abbreviations for individual amino acids. 
    N-terminal sequence: ELDEYLFSFG DGLYDAGNAK FIYPDKYLPS YHHPYGTTFF    
 
         [0011]     The amino acid sequence data is compared against the protein sequence database of the National Centre for Biotechnology Information (NCBI), USA, using the Basic Local Alignment Search Tool (BLAST) algorithm. The search revealed that the query sequences had good homology to plant GDSL-like lipases and myrosinase-associated proteins of crucifers.  
         [0000]     Preparation and Cloning of the cDNA Encoding the Smaller Components of Hev b4  
         [0012]     The complementary DNA (cDNA) encoding the amino acid sequence of the smaller components of Hev b 4 can be cloned and multiplied in a host such as a micro-organism. The micro-organism can be selected from the group consisting of bacteria, yeast, and viruses. Eukaryotic cells can also be used as vectors. The following is a description of the method used to clone the cDNA of the smaller components of Hev b 4. Standard procedures are used in the preparation and purification of DNA, agarose gel electrophoresis, ligations, transformations and total latex RNA isolation.  
         [0000]     Preparation of Latex Total RNA  
         [0013]     Latex is collected by tapping the  H. brasiliensis  tree. Before the tree is tapped, it is fitted with a sterilised drainage spout. Immediately upon tapping, the incision and spout are washed with about 20 ml of 2×RNA extraction buffer (0.1 M Tris-HCl, 0.3 M LiCl, 0.01 M EDTA, 10% SDS, pH 9.5). The latex is then washed down with 100 ml of 2×RNA extraction buffer to a total collected volume of 200 ml in a sterile conical flask. In the laboratory, the latex is mixed well and centrifuged in polyallomer tubes at 40,000 g for 30 minutes at 15° C. The aqueous phase is gently decanted into sterile centrifuge tubes and subsequent processing of the aqueous phase to isolate total RNA is performed according to the method of Prescott and Martin (1987).  
         [0000]     Synthesis of the cDNA Encoding the Smaller Components of Hev b 4  
         [0014]     First strand cDNA synthesis is prepared by reverse transcribing 2.5 μg of total RNA in a 25 μl reaction mixture using the 5′/3′ RACE™ Kit (Invitrogen, USA) as per vendor&#39;s instructions. Synthesis of double stranded cDNA is accomplished by PCR amplification.  
         [0015]     The complete cDNA sequence of the smaller components of Hev b 4 was obtained by combining both their 5′ and 3′ RACE products. An antisense degenerate primer designed based on conserved regions within plant GDSL-like lipases is used to initiate the first strand synthesis to capture the 5′ terminus. Oligo-dT primer is used to reverse transcribe the 3′ terminus of the transcript. An aliquot of the reverse transcription containing the 5′ terminus of the transcript is amplified in a thermocycler using the combination of the abridged anchor primer (supplied by the manufacturer) and antisense degenerate gene specific primer. Similarly, an aliquot of the reverse transcription containing the 3′ terminus of the transcript is amplified using the combination of oligo-dT and sense-strand gene specific primer. A second round of PCR is performed using 2 μl of the first round amplifications as the template. 10 μl of each secondary PCR is used for analysis on a 1.2% agarose gel containing ethidium bromide for detection of DNA.  
         [0000]     Cloning of the cDNA Encoding the Smaller Components of Hev b 4  
         [0016]     The 5′ and 3′ RACE products are ligated into pGEM-T® vector (Promega, USA) as per the vendor&#39;s instructions. The ligates (3 μl) are used for the transformation of the vector into  Escherichia coli  JM109 (Promega, USA). After incubating overnight at 37° C. in LB agar medium containing amplicillin (100 μg/ml) and Xgal (80 μg/ml)/IPTG (3 mmol/L), putative transformants that appear as white colonies were selected from the background of blue colonies. The picked clones are screened using the Wizards SV Minipreps DNA Purification System (Promega, USA) as per vendor&#39;s instructions. One μg plasmid DNA of the selected clones were then sent for nucleic acid sequencing.  
         [0017]     The 5′ and 3′ DNA sequences were combined and translated into amino acids that they encoded (see  FIGS. 1 and 2 ). The full sequence was elucidated for the cloned cDNA of 1375 base pairs. The predicted protein contained 366 amino acid residues that included a signal peptide of 24 amino acids. The calculated molecular weight of the mature protein is 38529.2 Da. There is a discrepancy between this molecular size (38.5 kDa) and the observed molecular size as indicated by SDS-polyacrylamide gel electrophresis (approximately 50 kDa). This difference is due to the carbohydrate component of the protein. The presence of the carbohydrate was confirmed using the GlycoTrack carbohydrate detection kit (Oxford Glycosystems, United Kingdom) following the manufacturer&#39;s instructions.  
         [0018]     A search in the protein domain database using the deduced amino acid sequences revealed the presence of lecithinase conserved domains. The deduced amino acid sequence data also encompassed the N-terminal segment obtained independently from protein sequencing. 
    gagctggatgaatatttgtttagctttggggatggactttatgatgcaggcaatgctaaatttatatatcctgataagtatctt ccctctatcaccatccatatggtaccactttcttc (at position 118 to 237 of the cDNA) encodes the peptide ELDEYLFSFG DGLYDAGNAK FIYPDKYLPS YHHPYGTTFF 
 
 where a,g,c and t are the abbreviations for the nucleotide bases adenine, guanine, cytosine and thymine respectively. The applicants note that minor variations of the DNA sequence would not alter substantially the basic characteristics of the peptide that the DNA encodes. 
 
 Demonstration of Protein Allergenicity 
   
 
         [0020]     A Western blot of the purified Hev b 4 protein complex was incubated with blood serum from latex allergic patients to determine if IgE (the immunoglobulin that mediates the allergic reaction) binds to the protein. Binding of IgE to the protein indicated that the protein is allergenic.  
         [0021]     Hev b 4 protein complex is separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% gels and transferred electrophoretically to a nitrocellulose membrane to obtain a Western Blot. The membrane is then pre-incubated with PBS containing 5% non-fat milk. To detect protein-IgE binding in Western blots the nitrocellulose membrane is incubated with serum of several latex-allergic patients (diluted 1:5.25 in PBS-milk and 0.05% sodium azide) as the primary antibody. Anti-human IgE conjugated to alkaline phosphatase served as the secondary antibody. The binding of human IgE to Hev b 4 on a Western blot of the protein after separation of the protein by SDS-polyacrylamide gel electrophoresis is shown in  FIG. 3 .  
         [0000]     Use of the Allergenic Protein Complex, Hev b 4 in Immunoassays  
         [0022]     Native or recombinant smaller components of Hev b 4 or its molecular variant (a protein similar to the smaller components of Hev b 4, but differing slightly, for example, in a few amino acids) can be used on its own, or in combination with an antibody developed against the smaller components of Hev b 4 or its molecular variant, in immunoassays. Molecular variants of the smaller components of Hev b 4 may occur naturally in latex or may exist as a result of laboratory manipulation. Such immunoassays can be constructed in many different formats, but they rely on the immunological reaction between an antibody and its antigen. The antibody in this instance can be human IgE or an antibody against the smaller components of Hev b 4, or its molecular variant. Its antigen can be native or recombinant smaller components of Hev b 4 or its molecular variant, or a peptide that embodies the epitope site of the protein or its molecular variant.  
         [0023]     Immunoassay can be used for the diagnosis of allergy to the smaller components of Hev b 4 or allergy to latex in general. In a different format, the immunoassay can be used for the detection of the smaller components of Hev b 4 in latex or latex products.  
         [0000]     Use of the Smaller Components of Hev b 4 in Immunotherapy  
         [0024]     Immunotherapy is a preventive treatment for allergic reactions that is carried out by giving gradually increasing doses of the allergen to which the person is allergic. The incremental increases of the allergen cause the immune system to become less sensitive to the substance when the substance is encountered in the future. There are several treatment protocols for immunotherapy. As an example, immunotherapy with the smaller components of Hev b 4 can be carried out by injecting the purified sample into skin of the arm. An injection may be given once a week for about 30 weeks, after which injections can be administered every two weeks. Eventually, injections can be given every four weeks. The duration of therapy may be three or four years, sometimes longer. In place of native smaller components of Hev b 4, immunotherapy may also be carried out with a suitable recombinant smaller components of Hev b 4, but differing slightly, for example, in a few amino acids), or a peptide representing a portion of the smaller components of Hev b 4 or its molecular variant.  
         [0025]     From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usage and conditions.  
       REFERENCES  
       [0000]    
       
          1. PRESCOTT, A AND MARTIN, C (1987). A rapid method for the quantitative assessment of levels of specific mRNAs in plants. Plant Mol. Biol. Reporter, 4, 219-224  
          2. SUNDERASAN, E., SAMSIDAR HAMZAH, SHARIFAH HAMID, WARD, M. A., YEANG, H. Y. AND CARDOSA, M. J. (1995) Latex B-Serum β-1,3-Glucanase (Hev b II) and a Component of the Microhelix (Hev b IV) are Major Latex allergens.  J. nat. Rubb. Res.,  10(2), 82-99  
          3. YEANG H. Y., SITI ARIJA M. ARIF, FARIDAH YUSOF, AND SUNDERASAN E. (2002). Allergenic proteins of natural rubber latex.  Methods  27, 32-45  
          4. SUNDERASAN E., WARD M. A., AND YEANG H. Y. (2002). Isolation and characterisation of  Hevea brasiliensis  latex cyanogenic glucosidase.  J. Rubb. Res.  5(4), 244-252  
          5. COHN, W. E. (1984) Nomenclature and symbolism of alpha-amino acids.  Methods Enzymo.  106,3-17.