Insect sting allergies to bees and vespids are common; the vespids include hornets, yellow jackets, and wasps(1, 2) . Susceptible people can be sensitized on exposure to minute amounts of venom proteins, as <10 µg of protein is injected into the skin on a single sting(3) . Insect venoms are used widely for the diagnosis and treatment of insect allergy(1, 2) . Patients are screened for the presence of specific IgEs by skin tests with venom and are protected against subsequent stings by subcutaneous injections of increasing amounts of venom. On treatment with venom, patients show a rapid rise of specific IgEs and IgGs followed by a gradual decrease of IgEs. Recent studies suggest that immunotherapy down-regulates allergen-specific T cell responses, thus altering the allergen-specific antibody response(4) .

Insect allergy, like other forms of immediate-type allergy, e.g. hay fever, results from interaction of cell-bound specific IgEs with allergens. Immune response, irrespective of the antibody isotype, is known to be controlled by the genetic make-up of the host, the route and the mode of immunization, and the nature of the immunogen. It is not clear to what extent the nature of the immunogen, or allergen, determines the extent of IgE response in susceptible people.

The vespids have similar venom compositions. They each contain a homologous set of three major venom allergens: antigen 5 of unknown biological function, hyaluronidase, and phospholipase A(5) . Antigens 5 from several species of different vespids have been cloned or sequenced and are found to be proteins of 203-205 amino acid residues(6) . Phospholipases A from white face hornet (Dolichovespula maculata) (7) and two species of yellow jackets (Vespula maculifrons and vulgaris)(8, 9) have been cloned or sequenced and are found to be proteins of 300 amino acid residues.

The enzymatic specificity of vespid hyaluronidase is of the endo-N-acetylhexosaminidase type(10) , as it catalyzes the release of reducing groups of N-acetylglucosamine from hyaluronic acid, a polymer of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine. In this paper we will report our findings on the cloning and expression of white face hornet hyaluronidase, its sequence similarity with the bee venom allergen hyaluronidase (11) and a mammalian testis protein known as PH-20(12, 13) , and its antigenic cross-reactivity with bee venom hyaluronidase. Human PH-20 was recently reported to have hyaluronidase activity(14) . The three venom proteins of white face hornet (D. maculata), antigen 5, phospholipase A, and hyaluronidase, are designated as Dol m 5, 1, and 2, respectively, according to an accepted nomenclature system for allergens(15) .

MATERIALS AND METHODS

Isolation and Partial Amino Acid Sequences of Hornet Hyaluronidase, Dol m 2

cDNA of Dol m 2

()

First strand cDNAs for 3`-RACE were synthesized from the MeHgOH denatured total RNAs (5 µg) as the template, with oligonucleotide number 5 (Table 1) as the primer, using the cDNA synthesis system from Life Technologies, Inc. For 5`-RACE, the single-stranded cDNAs were synthesized as described above, except that oligonucleotide number 8 (Table 1) was used as the primer, then poly(dA)-tailed with terminal deoxynucleotidyltransferase (U. S. Biochemical). 3`- or 5`-RACE was carried out with GenAmp PCR reagent kit (Perkin-Elmer Cetus) using Amplitaq polymerase. PCR was carried out for two successive rounds of 30 cycles as follows: template, 2 µM each sense and antisense primers, 400 µM each dNTPs, and 5 units of Taq polymerase (Perkin-Elmer Cetus) in 50 µl of 10 mM Tris-HCl (pH 8.4) + 50 mM KCl + 1.5 M MgCl. Each cycle consisted of 45 s at 94 °C, 25 s at 50 °C, and 3 min at 72 °C. The amount of template for the first round of PCR was equivalent to 50 ng of total RNAs used for cDNA synthesis, and that for the second round of PCR was 1/1000 of the first PCR reaction mixture.

PCR products were examined by electrophoresis in agarose gel with ethidium bromide staining and by Southern blot analysis where necessary. PCR products, which contain single 3`-overhanging A nucleotides(20) , were used for cloning into the pCR vector with compatible T-nucleotide overhangs (Invitrogen Corp.). Plasmid DNAs were isolated using the Wizard Miniprep system (Promega Biotec). DNA sequences were determined by the dideoxynucleotide chain termination method using alkaline-denatured plasmid DNAs and the Sequenase Version 2.0 kit (U. S. Biochemical).

Expression of r-Dol m 2 with Ile to Phe Mutation at Residue 47

After phenol-chloroform extraction and ethanol precipitation, the amplified cDNA (about 2 µg) was digested in 60 µl of universal buffer with 20 units each of restriction enzymes BamHI and BglII or HindIII (Life Technologies, Inc.) at 37 °C overnight. The product was isolated by electrophoresis in 1.4% LGT agarose (FMC). The gel slice containing the cDNA of interest was cut out, and 2-4 µl of the melted gel (40-80 fmol of cDNA) was used for ligation with 18 ng (8 fmol) of appropriately cut and dephosphorylated pQE-12 or -8 vector (QIAGEN). Ligation was carried out overnight at 15 °C in the presence of 2 units of T4 DNA ligase in 60 µl of buffer as supplied by the manufacturer (Life Technologies, Inc.).

Competent M15 [pREP4] cells were transformed with the above ligation mixture, then grown on LB-agar plates containing 25 µg/ml kanamycin and 100 µg/ml ampicillin as described by the manufacturer (QIAGEN). Selected colonies were screened by SDS-gel electrophoresis for expression of the expected protein after induction with 1 mM isopropyl-1-thio--D-galactopyranoside for 1.5 h.

Positive colonies were grown in a culture of 250-ml volume. The collected bacteria pellet was washed free of culture with buffer of 0.05 M Tris-HCl (pH 8.0) + 0.10 M NaCl + 1 mM EDTA, then dissolved in 6 ml of 6 M guanidine hydrochloride + 0.10 M Tris-HCl (pH 8.0) + 20 mM 2-mercaptoethanol and heated to 100 °C for 5 min. After clarification by high speed centrifugation, the solution was applied to a 0.8 3.5 cm column of nickel-nitrilotriacetic acid agarose (QIAGEN). The column was washed in succession with 6-ml portions of 6 M urea + 20 mM 2-mercaptoethanol buffered at pH 8.0, 6.3, 5.9, and 4.5. r-Dol m 2 and its fragments were eluted with 6 M urea buffered with 0.14 M NHAc at pH 4.5.

r-Proteins were freed of urea and buffer salts by chromatography on a 8 0.9 cm column of C18 silica (Separation Group, Hesperia, CA) with a linear 2-propanol gradient of 0.5% per ml in 0.1% trifluoroacetic acid. The r-proteins were eluted at about 40% 2-propanol. After lyophilization, they were found to be soluble in 0.02 N HOAc, and their concentrations were determined by absorbance at 280 nm. The molar extinction coefficients of proteins at 280 nm were calculated from their tyrosine and tryptophan contents, with values of 1280 and 5690, respectively.

Expression of r-Dol m 2 with Met to Leu Mutation at Residue 122

Full length cDNA encoding the mutated Dol m 2 was obtained by PCR amplification of a mixture of mutated cDNAs encoding 1-125 and 119-331 using primers 11 and 13. The resulting PCR product was digested with BamHI and HindIII, then ligated with appropriately cut and dephosphorylated pQE8. The remaining steps were the same as those described above.

Characterization of r-Dol m 2

Attempted Refolding of Recombinant Dol m 2

Immunization and Immunoassays

Antibodies were measured by enzyme-linked solid-phase immunoassay. Microtiter wells were coated overnight with 5 µg/ml antigen in 0.05 M Tris-HCl (pH 8.0). Any remaining reactive sites of wells were blocked with diluent buffer which contained 0.1 mg/ml bovine serum albumin, 0.5 mg/ml Tween 20, 0.5 M NaCl, and 0.05 M Tris-HCl (pH 8.0). The wells were then treated for 1-h periods in succession with varying concentrations of mouse sera to be tested, 10 µg/ml rabbit antibody specific for mouse IgGs, and 1/100 diluted goat antibody specific for rabbit IgG conjugated with horseradish peroxidase. Finally, the bound peroxidase was detected by absorbance at 490 nm after incubation with a substrate solution of 16 mg/ml phenol, 0.5 mg/ml 4-aminoantipyrine, and 0.005% HO in 0.1 M sodium phosphate (pH 7.0). Antibody titers were expressed as reciprocal dilutions required to give an absorbance change of 1.0 in 30 min.

Antigen and antibody solutions used above were prepared in diluent buffer. For enzyme-linked immunosorbent assay on solid-phase recombinant protein, the diluent buffer contained, in addition to the ingredients given earlier, 10% bacterial lysate. This lysate was prepared by sonication of bacteria in phosphate-buffered saline (containing 2 mM phenylmethanesulfonyl fluoride, 5 mM benzamidine, and 1 mM 2-mercaptoethanol) in 1/10 of the original culture volume, and the suspension was clarified by centrifugation before use. Addition of bacterial lysate was necessary to reduce background color development which might be due to the presence of antibodies specific for bacterial proteins in the reagents used.

Proliferation assays were made with spleen cells from two mice, 10 days after week 2 or week 8 immunizations. Spleen cells (2 10) were cultured with varying concentrations of test antigen in 0.2 ml of culture medium at 37 °C and 5% CO. [H]Thymidine (1 µCi) was added on day 3, and the uptake of thymidine was counted on day 4. The culture medium consisted of RPMI 1640 (Life Technologies, Inc.), 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES buffered at pH 7.3 (Sigma), and 54 µM 2-mercaptoethanol.

RESULTS

Cloning of Hornet Hyaluronidase, Dol m 2

From the DNA sequence data encoding residues 8-45 of hyaluronidase, additional oligonucleotide primers 3 and 4 (Table 1) were synthesized. They were used together with oligonucleotide primers 6 and 7 (Table 1) to amplify the 3` ends of the cDNA encoding hyaluronidase by the 3`-RACE procedure(18, 19) . In this manner, a cDNA fragment containing nucleotides 127-1229 (Fig. 1) was obtained. Another set of primers, 8-10 (Table 1), was synthesized based on the DNA sequence data of 3`-RACE. They were used together with primers 5 and 6 to amplify the 5` end of the cDNA, and the cDNA fragment containing nucleotides 1-245 was obtained.

Figure 1: cDNA and amino acid sequences of hornet hyaluronidase (Dol m 2). Nucleotide and amino acid positions are numbered on the right. Numbering of amino acid residues begins and ends at its N and C termini of serine and asparagine, corresponding to nucleotide positions of 61-63 and 1051-1053. The underlined amino acid sequence was established by Edman degradation. The GenBank accession number for the above sequence is L34548.

The N-terminal sequence of hyaluronidase for residues 1-45, which was deduced by Edman degradation, is encoded by nucleotide position 61-195 in Fig. 1. The region of nucleotide position 1-60 probably encodes a portion of the prepro segment of hyaluronidase. However, the presence of a stop codon at nucleotide position 19-21 is unexpected and may possibly represent incomplete splicing of mRNA. The coding region of the DNA in Fig. 1ends at position 1053, as a stop codon follows that position. The region of nucleotide position 1057-1229 represents the 3`-untranslated region with a poly(A) tail.

Oligonucleotide primers 11 and 12 (Table 1) were synthesized from the data in Fig. 1. They were used to amplify the cDNA encoding full length hyaluronidase, with flanking BamHI and BglII restriction sites at the 5` and 3` ends, respectively. The full length cDNA was used for expression in bacteria as will be described later.

For sequence analysis, all fragments were cloned into a plasmid vector. The DNA sequence in Fig. 1was assembled from the data of 5 clones from 3`-RACE, 4 clones from 5`-RACE and 1 clone from specific PCR for expression of hyaluronidase. There are sufficient overlaps of the sequence data of these clones such that every nucleotide position in Fig. 1represents the consensus of 4 or more clones. The only exception is the region of position 1-45, which was obtained from 2 clones. There are several mutations of these clones which are listed in Table 2. Most of them are silent mutations, but 2 of them result in amino acid substitutions. These mutations may be due to infidelity of base incorporation in PCR, or they may represent allelic forms.

The amino acid sequence from the DNA data in Fig. 1indicates that hyaluronidase has 331 amino acid residues with a molecular mass of 38,929 daltons, as compared to a molecular mass of about 42 kDa from SDS-gel electrophoretic data. The difference in molecular mass suggests that the natural hyaluronidase is a glycoprotein, as the translated sequence has a potential asparagine glycosylation motif of AsnX-Thr/Ser at residue 79-81.

Sequence Similarity of Hornet Hyaluronidase Dol m 2 with Other Proteins

Figure 2: Sequence comparison of honey bee and hornet venom hyaluronidases and human sperm protein PH-20. Alignment starts with residue 1 for both hyaluronidases and residue 40 for PH-20. Bee venom hyaluronidase and PH-20 contain 349 and 495 residues, respectively. Gaps, indicated by hyphens, were added to maximize sequence homology. Filled circles indicate residues of bee or human protein identical with those of hornet protein.

Expression of Hornet Hyaluronidase Dol m 2

The recombinant pQE8 or 12 plasmid was used to transform competent M15(pREP4) bacteria. On induction with isopropylthiogalactoside, recombinant proteins were expressed by the bacteria and could be isolated by metal ion chelation chromatography because of the presence of a hexahistidine tag. The expected sequence of the recombinant hyaluronidase (hya) from pQE8 is MRGSHGS-hya-KLN, and that from pQE12 is MRGS-hya-SRH. Their expected molecular masses are 40,539 and 40,340 daltons, respectively. On SDS-gel electrophoresis, the purified proteins from pQE8 contained mainly two bands of about 27 and 12 kDa (results not shown), and the purified proteins from pQE12 also contained two bands of about 40 and 28 kDa (Fig. 3A, lane 2). On immunoblotting, both bands from pQE12 expression bound hyaluronidase-specific mouse antisera (Fig. 3B, lane 2), but those from pQE8 expression did not (results not shown).

Figure 3: SDS-gel electrophoresis and immunoblot of natural and recombinant hornet hyaluronidase Dol m 2 in patterns A and B, respectively. Lane 1, natural protein; lane 2, recombinant protein with Ile to Phe mutation at residue 47 from pQE12 vector; and lane 3, recombinant protein with Met to Leu mutation at residue 122 from pQE8 vector.

Attempts to separate the 40- and 28-kDa proteins from pQE12 expression by various chromatographic means were unsuccessful. These two proteins were transferred from SDS gel by electroblotting onto Immobilon PSQ membrane, then sequenced by Edman degradation for 20 cycles. The 40-kDa protein contains the expected N-terminal sequence of Dol m 2 preceded by the tetrapeptide of MRGS. The size and the sequence data of the 40-kDa protein, as well as the immunoblot data, all suggest that it is the desired recombinant protein. The recombinant protein is probably acylated at its N terminus, as the yield of amino acid phenylthiohydantoin at each step of degradation is low for the 40-kDa protein when compared to that for the 28-kDa protein.

The 28-kDa protein contains a sequence corresponding to a fragment beginning at residue 123 of Dol m 2. To test whether the 28-kDa protein was generated by proteolysis of the recombinant Dol m 2, site-directed mutagenesis of the hyaluronidase cDNA was made by a PCR method (21) to replace the adenine base at position 424 with a cytosine. The resulting codon change yields a mutant with a Leu-Met bond in place of the natural Met-Met bond. Bacteria were transformed with recombinant pQE8 containing the mutant Dol m 2, and the desired mutant protein was expressed on induction with isopropylthiogalactoside. After purification by successive metal ion chelation and reverse phase chromatography, the mutant protein was isolated in a yield of about 2.5 mg/liter of culture. The purified mutant protein gave mainly one band of about 40 kDa on SDS-gel electrophoresis and immunoblotting (Fig. 3, A and B, lane 3). The immunoblot was detected with mouse sera specific for natural and recombinant Dol m 2 with identical results, but only the results with recombinant Dol m 2 specific sera are shown in the figure.

Hyaluronidase Activity of Recombinant Dol m 2

Immunological Properties of Recombinant Dol m 2

Data on antigen-stimulated proliferation of spleen cells from mice immunized with natural Dol m 2 or recombinant Dol m 2 with an Ile to Phe mutation are given in Fig. 4, A or B, respectively. In both graphs, lower concentrations of the natural protein were required for maximal proliferation than for the recombinant protein, and both graphs suggest similar maximal stimulations for both proteins. The lower concentration of the natural protein required for maximal stimulation may reflect its ease of uptake and/or processing by the appropriate cells for presentation to T cells. These data suggest that the recombinant and natural proteins have common T cell epitopes.

Figure 4: Proliferation assay with spleen cells from mice after 5 immunizations with natural hornet hyaluronidase and recombinant hornet hyaluronidase with Ile to Phe mutation at residue 47, in patterns A and B respectively. The stimulating antigens are the natural hornet protein (filled circles) and the recombinant protein with Ile to Phe mutations at residue 47 (open circles). Cell proliferation was measured by the uptake of [H]thymidine. Background proliferation was 3400 and 6000 cpm for patterns A and B, respectively. Error bars represent S.D. of triplicate measurements.

Antigen Cross-reactivity of Hornet and Bee Venom Hyaluronidases

Figure 5: Enzyme immunoassay of hornet and bee venom hyaluronidase-specific mouse sera on solid-phase hornet and bee hyaluronidases (patterns A and B, respectively). Hornet hyaluronidase-specific sera of week 5 and 7 bleedings are designated by open and closed circles, and those for bee-specific sera are designated by open and closed squares, respectively.

To study cross-reaction at the T cell level, proliferation assays with spleen cells from hornet or bee hyaluronidase immunized mice were carried out. Spleen cells from mice after 2 immunizations with hornet hyaluronidase at week 3 responded equally well on stimulation with hornet or bee hyaluronidase (Fig. 6A), and the magnitude of spleen cell response to hornet protein increased after 3 more immunizations with hornet protein at week 9, but the magnitude of response to bee protein remained about the same or decreased slightly (Fig. 6B). Similar studies showed that the spleen cells from mice after 2 immunizations with bee hyaluronidase at week 3 responded strongly on stimulation with bee protein and weakly with hornet protein (Fig. 6C); this difference persisted after 3 more immunizations at week 9 (Fig. 6D).

Figure 6: Proliferation assay with spleen cells from mice after 2 or 4 immunizations with hornet venom hyaluronidase (patterns A and B, respectively) and from mice after 2 and 4 immunizations with bee venom hyaluronidase (patterns C and D, respectively). The stimulating antigens are hornet hyaluronidase (filled circles) and bee hyaluronidase (filled squares). Cell proliferation was measured by the uptake of [H]thymidine. Background proliferation was 8000 ± 2000 cpm. Error bars represent S.D. of triplicate measurements.

DISCUSSION

In Table 3are listed the known allergens of honey bee and white face hornet venoms, of which amino acid sequences have been determined by chemical sequencing or by molecular cloning. Bee venom has three allergens: hyaluronidase, phospholipase A, and melittin, which is a 26-residue peptide. Hornet venom also has three allergens: antigen 5, hyaluronidase, and phospholipase A. The other vespids, yellow jackets and wasps, have allergens homologous to those of hornets.

As shown in Fig. 2, there is 56% sequence identity of hornet and bee hyaluronidases. The sequence identity of hornet and bee hyaluronidases raises the possibility that they may have common or cross-reacting B and T cell epitopes. This is supported by the immunoassay data with antibodies from mice immunized with hornet or bee hyaluronidase (Fig. 5) and by the proliferative assay with spleen cells from these mice (Fig. 6). The antigenic cross-reactivity of yellow jacket and bee hyaluronidases has been detected by RAST inhibition assay with sera from allergic patients(32, 33) . These findings together may explain the common observation that the majority of insect allergic patients exhibit multiple sensitivity to bees and vespids(1, 2) . Thus, the observed multiple sensitivity can be a consequence of multiple exposures to different insects and/or the cross-reactivity of bee and vespid hyaluronidases.

In addition to the insect venom allergens, the amino acid sequences of 70 or more allergens from pollen, mites, animal danders, molds, foods, etc. are known (cf. (15) ). These allergens have different sequences and biological functions. One common feature of these allergens is their varying extents of sequence identity with other proteins in our environment. This is exemplified by the data in Table 3for insect venom allergens. Each venom allergen in Table 3has at least 20% sequence identity with a mammalian protein. In each case there is more than one region of sequence identity ranging in size from penta- to longer length peptides. These regions of sequence identity can function as common B or T cell epitopes, since the minimal sizes of B and T cell epitopes are in the range of penta- to decapeptides.

Normally, a host does not mount immune responses to self-proteins. It is possible that insect allergic patients recognize their self proteins, so that they are primed to respond to the cross-reacting venom allergens. This would explain why susceptible people can become sensitized readily on exposure to microgram amounts of allergen. This hypothesis remains to be proven.

It has been reported that in people with a high total IgE level, positive skin venom tests were more frequent in males than in females (34) . Two of the venom allergens in Table 3have sequence identity with mammalian testis proteins. Vespid antigen 5s have sequence identity with a mammalian testis protein, and hornet and bee hyaluronidases have sequence identity with another testis protein. When hornet antigen 5 was tested in male and female mice of BALB/c strain, no difference in immunogenicity for IgG antibody or for T cell response was observed in male or female animals. ()

One problem in immunotherapy of patients is the limited dose of allergens which can be administered safely(1, 2, 4) . Recent model experiments showed that treatment of mice with the dominant T cell epitopes of the major cat or mite allergen (35, 36) induced suppression of allergen-specific immune response. Although the exact mechanism of the reported immunosuppression is still under study(37, 38) , these findings are of interest. Peptides are less likely to induce systemic reactions in patients than multivalent allergens with their full complement of B cell epitopes of the continuous and discontinuous types.

The present work on recombinant hornet hyaluronidase, as well as our published report (39) on recombinant hornet antigen 5 and our unpublished results on recombinant hornet phospholipase A, is relevant to studies on the possible use of peptide fragments as immunotherapeutic reagents. All three recombinant proteins lack the native conformation of the natural proteins, as indicated by the lack of enzymatic activity where applicable, and by the absence of the discontinuous type B cell epitopes of the natural proteins. Nonetheless, they and their fragments retain the T cell epitopes of the natural proteins, as T cell epitopes consist only of sequentially adjacent amino acid residues(40) .

FOOTNOTES

*

This research is supported in part by Grant AI-17021. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34548[GenBank].

()

The abbreviations used are: RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; r, recombinant.

()

T. P. King, unpublished results.

ACKNOWLEDGEMENTS

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