Mutagenic and chemical modification of the ABA-1 allergen of the nematode Ascaris: Consequences for structure and lipid binding properties

The polyprotein allergens/antigens of nematodes (NPAs) are the only lipid b inding proteins known to be produced as polyproteins. Cleavage of the large polyprotein precursors at regularly spaced proteinase cleavage sites produ ces 10 or 11 individual protein units of similar to 15 kDa. The sequences o f these units are highly diverse within and between species, but there are five absolutely or strongly conserved amino acid positions (Trp 15, Gln20, Leu42, Cys64, and Cys120). We have tested the role of these signature amino acids by mutational or chemical alteration of the ABA-1 protein of Ascaris , and examined the resulting modified proteins for perturbations of their l ipid binding activities and structural integrity. Substitution of Trp15 and Gln20 both affect the stability of the protein in terms of resistance to t hermal or chemical denaturation, but the ligand binding function is unaffec ted. Mutation of Leu42, however, disrupts both the protein's structural sta bility and functional integrity, as does chemical disruption of the disulfi de bridge formed between Cys64 and Cys120. We also find that the C-terminal , but not the N-terminal, half of the protein binds fatty acids, indicating that the binding site may be confined to this part of the protein. This al so supports the idea that the NPA units are themselves derived from an anci ent duplication event, and that they may comprise two functionally distinct domains.