Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

Abstract

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein.The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holoallergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4 1 22 with cell dimensions of a ‫؍‬ 87.7 Å, b ‫؍‬ 87.7 Å, and c ‫؍‬ 59.6 Å.We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

The Major Grass Pollen Group 5 Allergen from <i>Dactylis glomerata </i>and Its C-Terminal Split Product Both Behave as Dimers: Implications for Allergen Standardization

“…The 25-kDa fraction had an apparent M r of 12 kDa and was thought (but not confirmed) to be a degradation product [5]. Another indication for the existence of a group 5 degradation product was reported by Bufe et al [23]. They observed the spontaneous appearance of a C-terminal peptide of around 12 kDa after Phl p 5b expression in Escherichia coli.…”

“…Whether the C-terminus of Dac g 5 is generated posttranslationally, by proteolytic activity, or whether the peptide has its own gene is not known. So far, there are no reports showing that the natural allergen is degraded to its C-terminal half, except for two recombinant Phl p 5 products [23, 45]. Maglio et al [45]reported a time-dependent degradation of the N-terminus of rPhl p 5a.…”

“…For recombinant Phl p 5, Bufe et al [22, 23]reported that the C-terminal half of the allergen appears spontaneously after expression in Escherichia coli , and that this peptide has RNAse activity. Whether the C-terminus of Dac g 5 is generated posttranslationally, by proteolytic activity, or whether the peptide has its own gene is not known.…”

“…From X-ray crystallographic analysis it was concluded that this C-terminal peptide crystallized as a dimer. Upon resolubilization, this dimer showed similar allergenicity compared with the whole allergen [23, 24]. Recently, this C-terminal dimer was shown to consist of four α-helices.…”

“…The authors also pointed to significant internal homology between the C- and the N-terminal halves of Phl p 5, and suggested similar structural features for both halves, possibly facilitating dimerization. Bufe et al [23]have also demonstrated that IgE antibodies affinity-purified from the C-terminal peptide also recognize the N-terminal half of the molecule [24]. …”