Comparison of natural and recombinant isoforms of grass pollen allergens.
More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in 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 allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively. By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens. The post-translational modifications (one N-glycosylation 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 high IgE cross-reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.
Petersen
A
A
Forschungszentrum Borstel, Germany.
Grobe
K
K
Lindner
B
B
Schlaak
M
M
Becker
W M
WM
D000485:Allergens / Q000032:analysis*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics; D006109:Poaceae / Q000737:chemistry*; D011058:Pollen / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D052179:Antigens, Plant; D006023:Glycoproteins; C100985:HOL L 1 protein, Holcus lanatus; C042814:Lol p I protein, Lolium perenne; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C090396:PHLPI protein, Phleum pratense