• allergen;
  • antigenic binding sites;
  • baker's yeast;
  • house-dust mite;
  • IgE;
  • monoclonal antibodies;
  • recombinant Der p 2;
  • Saccharomyces cerevisiaer, site-directed mutagenesis


  1. Top of page
  2. Abstract
  3. References

Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae. was used as a tool to determitie IgE- and monoclonal antibody (mAb)-binding sites on this allergen. For this purpose, mutant molecules were produced by application of site-directed mutagenesis. The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues in three predicted antigenic sites (residues 45–48, 67-1-69. and 88–90) were replaced by alanine residues. IgE- and mAb reactivity to these mutants was compared to that to “wild type” Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb aDpX. Mutations in the areas adjacent to this loop (i.e. 67–69 and 88–90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure. The area of residues 45–48 was shown to be involved in an epitope for mAb 2B12. The reactivity of mAb 7A1 was influenced by substitutions of residues 45–48 as well as 88–90. Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (lOEll, αDpX. and 7A1). From these observations, it may be concluded that binding of different mAbs is influenced by the same mutations and that the binding of single mAbs is influenced by two or more mutations scattered over the allergen molecule. These findings can point in two directions: minor aminoacid changes result in disruption of the overall conformation of the allergen, or distant sites are close together in the three-dimensional structure of the allergen. Decreased IgE reactivity was observed with all mutant molecules, varying between patients. The observed effects ranged from 5- to 1000-fold. Deletion of the amino-acid residues spanning cys21-cys27 and cys73-cys78 had the strongest effect on IgE reactivity, where decreases up to 1000-fold were observed. Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.




monoclonal antibody


phosphate-buffered saline


PBS+0.1% (v/v) Tween-20


PBS-T+0.3% (w/v) BSA


radioallergosorbent test




RAST unit


wild type


  1. Top of page
  2. Abstract
  3. References
  • 1
    Chua KY, Doyle CR, Simpson RJ, Turner KJ, Stewart GA, Thomas WR. Isolation of cDNA coding for the major mite allergen Der p 2 by IgE plaque immunoassay Int Arch Allergy Appl Immunol 1990;91:11823.
  • 2
    Nishiyama C, Yuuki T, Takai T, Okumura Y, Okudaira H. Determination of three disulfide bonds in a major house dust mite allergen Der f2. Int Arch Allergy Appl Immunol 1993;100:15966.
  • 3
    Chua KY, Green WK, Kehal P, Thomas WR. IgE binding studies with large peptides expressed from Der p 2 cDNA constructs. Clin Exp Allergy 1991;21:1616.
  • 4
    van ‘t Hof W, Driedijk PC, van den Berg M, Beck-Sickinger AG, Jung G, Aalberse RC. Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p 2 using overlapping synthetic peptides. Mol Immunol 1991;28:122532.
  • 5
    Atassi H, Atassi MZ. Antibody recognition of ragweed allergen Ra3; localization of the full profile of the continuous antigenic sites by synthetic overlapping peptides representing the entire protein chain. Eur J Immunol 1986;16:22935.
  • 6
    Mazur G, Bauer X, Modrow S, Becker WM. A common epitope on major allergens from non-biting midges (Chironomida). Mol Immunol 1988;25:100510.
  • 7
    Elsayed S, Titlestad K, Apold J, Aas K. A synthetic hexapeptide derived from allergen M imposing allergenic and antigenic reactivity. Scand J Immunol 1980;12:1715.
  • 8
    van Milligen FJ, van 't Hof W, van den Berg M, Aalberse RC. IgE epitopes on the cat (Felis domesticus) major allergen Fel d I: a study with overlapping synthetic peptides. J Allergy Clin Immunol 1994;93:3443.
  • 9
    Elsayed S, Holen E, Dybendal T. Synthetic allergenic epitopes from the amino-terminal regions of the major allergens of hazel and birch pollen. Int Arch Allergy Appl Immunol 1989;89:41015.
  • 10
    Lombardero M, Heymann P, Platts-Mills T, Fox J, Chapman M. Conformational stability of B cell epitopes on group I and group II Dermatophagoides spp. allergens: effect of thermal and chemical denaturation on the binding of murine IgG and human IgE antibodies. J Immunol 1990;144:135360.
  • 11
    Hakkaart GAJ, Harmsen MM, Chua KY, Thomas WR, Aalberse RC, van Ree R. Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae. Clin Exp Allergy 1997 (in press).
  • 12
    Smith AM, Chapman MD. Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2. Mol Immunol 1996;33:399405.
  • 13
    Nishiyama C, Fukada M, Usui Y, et al. Analysis of the IgEepitope of Der f 2, a major mite allergen, by in vitro mutagenesis. Mol Immunol 1995;32:10219.
  • 14
    Chou PY, Fasman GD. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry 1974;13:21122.
  • 15
    Chou PY, Fasman GD. Prediction of protein conformation. Biochemistry 1974;13:21245.
  • 16
    Chou PY, Fasman GD. Empirical predictions of protein conformations. Ann Rev Biochem 1978;47:25176.
  • 17
    Chou PY, Fasman GD. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 1978;47:45148.
  • 18
    Robson B, Suzuki E. Conformational properties of amino acid residues in globular proteins. J Mol iol 1976;107:32756.
  • 19
    Garnier J, Osguthorpe DJ, Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 1978;120:97120.
  • 20
    Jameson BA, Wolf H. The antigenic index: a novel algorithm, for predicting antigenic determinants. Comput Appl Biosci 1988;4:1816.
  • 21
    van der Zee JS, van Swieten P, Jansen HM, Aalberse RC. Skin test and histamine release with PI-depleted Dermatophagoides pteronyssinus body extracts and purified P1. J Allergy Clin Immunol 1988;81:88496.
  • 22
    Ovsyannikova IG, Vailes LD, Li Y, Heymann PW, Chapman MD. Monoclonal antibodies to group 2 Dermatophagoides spp allergens: murine immune response, epitope analysis and development of a two site ELISA. J Allergy Clin Immunol 1994;94:53746.
  • 23
    Heymann PW, Chapman MD, Aalberse RC, Fox JW, Platts-Mills TAE. Antigenic and structural analysis of group II allergens (Der f II and Der p II) from house dust mites (Dermatophagoides spp). J Allergy Clin Immunol 1989;83:105567.
  • 24
    Jones EW. Proteinase mutants of Saccharomyces cerevisiae. Genetics 1977;85:2333.
  • 25
    Klebe RJ, Harris JV, Sharp ZD, Douglas MG. A general method for poly-ethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 1983;25:33341.
  • 26
    Takai T, Yokota T, Yasue M, et al. Engineering of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy. Nat Biotech 1997;15:7548.