N-terminus Amino Acid Analysis Of Four Independently Produced Recombinant Alt a1

RATIONALE: The Alternaria allergen, Alt a 1, is a major allergen. In previous work, we compared the IgE binding capacity of recombinant Alt a 1(rAlt a1) allergens. In this study, we examined the N-terminal amino acid (AA) sequences of the rAlt a1 proteins to determine if specific AA substitutions in the molecules (isoforms) were related to variations in IgE binding. METHODS: Recombinant Alt a1 samples were obtained from 4 sources, including our own laboratory. The samples were separated by SDS-PAGE, transferred to PVDF membranes, and the membranes stained with amido black. Protein bands corresponding to the molecular weight of rAlt a1 16 Kd were cut from the membrances and analyzed for their AA sequences by the Edman Degradation procedure. IgE binding to the rAlt a1 samples was determined by immunoblotting. RESULTS: We found a highly conserved region of AA sequences beginning at position 30 from the N-terminus. A substitution from glutamic acid to lysine at position 36 from the N-terminus was associated with less IgE binding in one sample. Another sample that had methionine at position 25 from the N -terminus, which was not present in the other samples, exhibited more intense IgE binding on the immunoblot. CONCLUSIONS: Amino acid changes in isoforms of r Alt a1 molecules and the methods by which these recombinant allergens are produced either in bacteria or yeast may affect their biological activity.