title: Mutagenic and chemical modification of the ABA-1 allergen of the nematode Ascaris: consequences for structure and lipid binding properties.
abstract: The polyprotein allergens/antigens of nematodes (NPAs) are the only lipid binding proteins known to be produced as polyproteins. Cleavage of the large polyprotein precursors at regularly spaced proteinase cleavage sites produces 10 or 11 individual protein units of approximately 15 kDa. The sequences of these units are highly diverse within and between species, but there are five absolutely or strongly conserved amino acid positions (Trp15, 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 lipid binding activities and structural integrity. Substitution of Trp15 and Gln20 both affect the stability of the protein in terms of resistance to thermal or chemical denaturation, but the ligand binding function is unaffected. Mutation of Leu42, however, disrupts both the protein's structural stability and functional integrity, as does chemical disruption of the disulfide 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 also supports the idea that the NPA units are themselves derived from an ancient duplication event, and that they may comprise two functionally distinct domains.
authors.affiliation: Department of Chemistry and the Divisions of Infection and Immunity and of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland.
authors.forename: L
authors.initials: L
authors.lastname: McDermott
authors.forename: J
authors.initials: J
authors.lastname: Moore
authors.forename: A
authors.initials: A
authors.lastname: Brass
authors.forename: N C
authors.initials: NC
authors.lastname: Price
authors.forename: S M
authors.initials: SM
authors.lastname: Kelly
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.forename: M W
authors.initials: MW
authors.lastname: Kennedy
mesh_terms: D000485:Allergens; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D052179:Antigens, Plant; D001200:Ascaris; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry*; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: C093689:ABA-1 protein, Ascaris suum; D000485:Allergens; D000596:Amino Acids; D052179:Antigens, Plant; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D014364:Tryptophan; D005982:Glutathione Transferase; D003545:Cysteine