β-Lactoglobulin mutant Lys69Asn has attenuated IgE and increased retinol binding activity.
β-Lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids, fatty acids, drugs, and vitamins. β-LG also is one of the major allergens in milk. Despite a lot of research on decreasing cow's milk allergenicity, the effects of mutations of β-LG on recognition by IgE from cow's milk allergy (CMA) patients have not been investigated. We describe here the expression in the yeast Pichia pastoris of a mutant bovine β-LG, in which lysine at position 69, in the main epitopes of the protein, was changed into asparagine (Lys69Asn). The purity and native like folded structure of the recombinant Lys69Asn β-LG was confirmed by HPLC, SDS-PAGE, mass spectrometry and circular dichroism. Lys69Asn β-LG has a fourfold stronger affinity than the wild-type protein for retinol, palmitic acid, and resveratrol, as determined by quenching of the intrinsic tryptophan fluorescence. At the same time the Lys69Asn mutant had a 9 fold attenuated, compared with the wild-type, affinity for IgE of sera from patients suffering from cow's milk allergy, whereas no difference could be detected between mutant and wild-type for binding of the IgGs of four monoclonal antibodies. The results of this study demonstrated the significant role of Lys69 residue on the binding and immuoreactivity properties of β-LG.
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, 81746-73441, Iran. Electronic address: a.taheri@ast.ui.ac.ir.
Asghar
A
Taheri-Kafrani
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, 81746-73441, Iran.
Neda
N
Tavakkoli Koupaie
UR 1268, INRA BIA-FIPL, B.P. 71627, 44316 Nantes Cedex 03, France.
Thomas
T
Haertlé
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D007782:Lactoglobulins / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D016269:Milk Hypersensitivity / Q000097:blood / Q000276:immunology; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007782:Lactoglobulins; D013267:Stilbenes; D014801:Vitamin A; D019308:Palmitic Acid; D007073:Immunoglobulin E; D001216:Asparagine; D008239:Lysine; D000077185:Resveratrol
Competitive ELISA; Cow’s milk allergy; Immunoreactivity; Ligand binding; Mutation; β-Lactoglobulin