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Keywords:

  • Hev b 10;
  • latex allergy;
  • MnSOD;
  • recombinant allergen

Manganese-superoxide dismutase (MnSOD) of Hevea brasiliensis has been identified as an IgE-binding protein in natural rubber latex (1) and was designated as latex allergen Hev b 10 (2). Since its allergenicity has not been studied so far, we produced a recombinant maltose-binding protein (MBP), latex-MnSOD(rHev b 10) fusion protein, to investigate the IgE-binding reactivity of Hev b 10 with sera from latex-allergic patients.

cDNA (2 ng) from H. brasiliensis leaves (3) was amplified in the presence of 25 pmol modified Hev b 10-specific primers (5′-Hev b 10: 5′-GTATACGTACAGACCTTCT-CTCTCCCCGATCTC-3′; 3′-Hev b 10: 5′-CCGGAAATTCTAAGAAGAAGGGCATTCTTTGGC-3′) based on the nucleotide sequence of EMBL accession no. AJ249148 (partial mRNA of H. brasiliensis MnSOD), 50 mM KCl, 10 mM Tris, pH 9.0, 2.0 mM MgCl2, 0.01% (w/v) gelatin, 500 µM dNTP, and 2.5 units Taq polymerase at 95°C for 3 min, followed by 40 cycles (95°C, 1 min; 67°C, 1 min; 72°C, 1 min) and a final elongation at 72°C for 10 min (final volume: 50 µl; MWG Primus 25 cycler). The PCR product (635 bp) was adapted with Sna BI-Eco RI and subcloned into the XmnI-Eco RI digested pMAL-c2 (3). The resulting clone pMALcHev b 10 overexpressed a 65.6-kDa MBP-Hev b 10 fusion protein in E. coli host TG-1. Comparison of our Hevea MnSOD nucleotide sequence (EMBL accession no. AJ289158) with the sequence described above (accession no. AJ249148) revealed differences in three amino-acid positions (120: GCT [Ala][RIGHTWARDS ARROW]GTT [Val], 123: CGA [Arg][RIGHTWARDS ARROW]CAA [Glu], 147: GTA [Val][RIGHTWARDS ARROW]GTG [Val]).

The expression and purification of the fusion protein was performed as described recently for MBP-rHev b 1 (3). To investigate the IgE-binding reactivity, the MBP-rHev b 10 fusion protein was coupled onto nitrocellulose disks and was tested by enzyme allergosorbent test (EAST) (4) with sera from 20 latex-allergic health-care workers (HCWs) and from 20 latex-sensitive children with spina bifida (SB). Hev b 10-specific IgE values of >0.35 kU/l were considered positive. Five sera from patients without latex sensitization were used as controls. The results of the EAST showed that only two sera (1.27 kU/l and 0.43 kU/l) from 20 SB patients tested showed specific IgE antibodies to recombinant latex MnSOD. The 20 HCW sera and all controls including MBP revealed negative results. The specific IgE-binding to MnSOD was confirmed by immunoblotting for one serum sample (1:5 dilution) with MBP-MnSOD as antigen (Fig. 1; lane 2) and MBP as a control (Fig. 1, lane 1).

This patient also had specific IgE antibodies to latex (36 kU/l), Hev b 1 (3.89 kU/l), and hevein (Hev b 6.02; 3.58 kU/l). Furthermore, Aspergillus fumigatus-specific IgE antibodies were also detectable (5.76 kU/l). Since IgE to A. fumigatus may cross-react with various MnSODs (5), it cannot be excluded that the IgE-binding to MnSOD in this SB patient may have been caused by the cross-reactivity between A. fumigatus and MnSOD.

References

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  2. References
  • 1
    Posch A, Chen Z, Wheeler C, Dunn MJ, Raulf-Heimsoth M, Baur X. Charac-terization and identification of latex allergens by two-dimensional electro-phoresis and protein microsequencing. J Allergy Clin Immunol 1997;99:385395.
  • 2
    IUIS Allergen List. ftp://biobase.dk/pub/who-iuis/allergen.list.
  • 3
    Rihs HP, Chen Z, Schumacher S, et al. Recombinant Hev b 1: large scale production and immunological characterization. Clin Exp Allergy 2000;30:12851292.DOI: 10.1046/j.1365-2222.2000.00870.x
  • 4
    Chen Z, Cremer R, Posch A, Raulf-Heimsoth, Rihs HP, Baur X. On the allergenicity of Hev b 1 among health care workers and patients with spina bifida allergic to natural rubber latex. J Allergy Clin Immunol 1997;100:684693.
  • 5
    Mayer C, Hemmann S, Faith A, Blaser K, Crameri R. Cloning, production, characterization and IgE cross-reactivity to different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus. Int Arch Allergy Immunol 1997;113:213215.