COMPARISON OF NATURAL AND RECOMBINANT ISOFORMS OF GRASS-POLLEN ALLERGENS

Citation
A. Petersen et al., COMPARISON OF NATURAL AND RECOMBINANT ISOFORMS OF GRASS-POLLEN ALLERGENS, Electrophoresis, 18(5), 1997, pp. 819-825
Citations number
28
Categorie Soggetti
Biochemical Research Methods
Journal title
ISSN journal
01730835
Volume
18
Issue
5
Year of publication
1997
Pages
819 - 825
Database
ISI
SICI code
0173-0835(1997)18:5<819:CONARI>2.0.ZU;2-8
Abstract
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.