More than 95% of grass pollen allergic patients possess IgE antibodies
against grass group I, a heterogeneous group of glycoproteins found i
n all temperate grasses. We studied the structural variability of the
group I allergens in single species and among different grasses. By 2-
DE blotting using patients' IgE and monoclonal antibodies, we detected
IgE-reactive isoforms with molecular masses between 32 and 37 kDa and
focusing in a wide pI ranging from 4.7 to 7.6. While the group I alle
rgens of timothy grass (Phl p 1) were composed of 37 and 35 kDa compon
ents, only single isoforms were found for ryegrass (Lol p 1) and velve
t grass (Hol 1 1): 32 and 34 kDa, respectively. By IV-terminal microse
quencing we determined single amino acid substitutions in different-si
zed group I allergens. The post-translational modifications (one N-gly
cosylation site, two hydroxylated proline residues and seven cysteine
residues for potential disulfide formations), which contribute to IgE
reactivity, were identical in all. From the cDNA sequences we deduced
protein sequence homologies > 90%, a result which might explain the hi
gh IgE cross-reactivity among the grasses. In order to test whether re
combinant group I grass allergens can act as substitutes for the natur
al forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE i
mmunoblotting again demonstrated a microheterogeneity in molecular mas
s and pI. While the E. coli products were free from post-translational
modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein f
raction with a carbohydrate content of about 15 %. This rPhl p 1 is hy
perglycosylated compared to the nPhl p 1, which only has a 5 % carbohy
drate content.