PRELIMINARY STRUCTURAL STUDIES ON PHL P 5, A MAJOR GRASS POLLEN ALLERGEN

Kåre Meno¹, Michael D. Spangfort², Henrik Ipsen², Margit Wissenbach² and Michael Gajhede¹

¹Protein Structure Group, Dept. of Chemistry, University of Copenhagen, Denmark ²ALK-ABELLÓ Group, Bøge Allé 10-12, DK-2970 Hørsholm, Denmark

Keywords: grass pollen, allergy, allergen, structure, X-ray crystallography

Grass pollen allergens are the cause of allergic diseases in a significant part of the populations in Northern Europe and North America. Several grass pollen allergens have been identified and these are divided into groups on the basis of their physical and chemical properties [1]. Group 5 allergens are particularly important since most of the grass pollen sensitised individuals produce IgE antibodies that recognise these proteins [2]. The amino acid composition of the group 5 allergens from Phleum pratense pollen (Phl p 5) is unique with a very high alanine content (26 mol%), 1.4 mol% of the modified amino acid hydroxy-proline and no cysteines. The Phl p 5 group therefore shows no similarities to the amino acid composition of other known allergens [3].

The purpose of this study is to determine the structure of this clinically relevant protein, isolated and in complex with Fab fragments, by means of X-ray crystallography. This would permit structural analysis of the allergen and of the Fab-Phl p 5 complex. It would furthermore yield structural information concerning the physical, chemical and conformational properties of allergen epitopes, which have never been described previously.

A well performing recombinant expression system for Phl p 5 has been constructed, whereby sufficient amounts of the protein has be purified (as described for Bet v 1 [4]). Crystals of Phl p 5 isolated and in complex with a monoclonal murine IgG Fab fragment have been grown. Both crystal forms belong to the monoclinic space group C2 and they have the following cell dimensions:

a = 78.2 Å, b = 64.8 Å, c = 51.3 Å and b = 111°

a = 343 Å, b = 131 Å, c = 185 Å and b = 122°

respectively. The Phl p 5 crystals diffracted to 2.4 Å and a native data set has been obtained. The Fab-Phl p 5 crystals diffracted to 10 Å on the in house rotating anode X-ray equipment, but the powerful synchrotron at Lund, Sweden improved this substantially to 4.2 Å.

Preparation of heavy atom derivatives is in progress so phases can be obtained. The above results together with the newest progress in the structure solution will be presented on the poster.

1. King, T.P., Hoffman, D., Løwenstein, H., Marsh, D.G., Platts-Mills, T.A. & Thomas, W. (1994) Allergen nomenclature. Int. Arch. Allergy Immunol. 105, 224-233.
2. Matthiesen, F. & Løwenstein, H. (1991) Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses. Clin. Exp. Allergy 21, 309-320.
3. Matthiesen, F. & Løwenstein, H. (1991) Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V. Clin. Exp. Allergy 21, 297-307.
4. Spangfort, M.D., Ipsen, H., Sparholt, S.H., Aasmul-Olsen, S., Larsen, M.R., Mortz, E., Roepstorff, P. & Larsen, J.N. (1996) Characterization of purified recombinant Bet v 1 with authentic N- terminus, cloned in fusion with maltose-binding protein. Protein Expr. Purif. 8, 365-373.