Engineered Alt a 13 fragment of Alternaria alternata abrogated IgE binding without affecting T-cell stimulation.
BACKGROUND
Epitopes were delineated for allergenic proteins, but studies are required to identify residues mediating IgE binding. In the present study, the in silico approach was used to identify IgE-binding residues of Alt a 13(1-50) fragment and confirmed by experimental approach.


RESULTS
IgE-binding epitopes of Alt a 13 mapped computationally were cloned, expressed, purified, and characterized using various immunochemical and biophysical methods. Among four fragments of Alt a 13, Alt a 13(1-50) demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 13(1-50)-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 13(1-50)-T21F_N27I, caused reduced IgE binding, histamine release, and low IL-4 release on stimulation of Alternaria alternata positive patients peripheral blood mononuclear cells in vitro.


CONCLUSIONS
This suggests that residues T21 and N27 are important for the secondary structure. In conclusion, Alt a 13(1-50)-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.
Shankar
Jay
J
Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India.
Singh
Bhanu P
BP
Gaur
Shailendra N
SN
Arora
Naveen
N
D000293:Adolescent; D000328:Adult; D000528:Alternaria / Q000276:immunology*; D000908:Antibodies, Fungal / Q000097:blood; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D019295:Computational Biology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005260:Female; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D012882:Skin Tests; D013601:T-Lymphocytes / Q000276:immunology*; D055815:Young Adult
D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D000939:Epitopes; D050505:Mutant Proteins; D010446:Peptide Fragments; D007073:Immunoglobulin E