Background and objective Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM-allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine β-casein (βcap) without cross-reacting with bovine β-casein (βbov) despite a sequence identity of 91%. In the present study, we investigated the non-cross-reactive IgE-binding epitopes of βcap. Methods Recombinant βcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the reco... More
Background and objective Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM-allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine β-casein (βcap) without cross-reacting with bovine β-casein (βbov) despite a sequence identity of 91%. In the present study, we investigated the non-cross-reactive IgE-binding epitopes of βcap. Methods Recombinant βcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified βcap by IgE antibodies from 11 GM-allergic/CM-tolerant patients and 11 CM-allergic patients or by monoclonal antibodies (mAb) raised against caprine caseins. The allergenic potency of modified βcap was finally assessed by degranulation tests of humanized rat basophil leukemia (RBL)-SX38 cells. Results Non-cross-reactive epitopes of βcap were found in domains 44-88 and 130-178. The substitutions A55T/T63P/L75P and P148H/S152P induced the greatest decrease of IgE-reactivity of GM-allergic/CM-tolerant patients toward βcap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of βcap by specific mAb which could discriminate between βcap and βbov. The modified βcap containing the five substitutions was then unable to trigger the degranulation of RBL-SX38 cells passively sensitized with IgE antibodies from GM-allergic/CM-tolerant patients. Conclusions Although IgE-binding epitopes are spread all over βcap, a non-cross-linking version of βcap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants.
