SECRETION OF RECOMBINANT HUMAN IGE-FC BY MAMMALIAN-CELLS AND BIOLOGICAL-ACTIVITY OF GLYCOSYLATION SITE MUTANTS

We have constructed an expression vector that leads to secretion of th e whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more hom ogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosyla ted in Chinese hamster ovary cells. Both the double mutant and wild-ty pe hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (K-a in the range 10(10)-10(11 ) M(-1)), and were able to sensitize isolated human basophils for anti -IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity re ceptor, Fc epsilon RII (K-a = 7.3 x 10(7) M(-1)), whereas the wild-typ e hIgE-Fc bound with a 10-fold lower affinity (K-a = 4.1 x 10(6) M(-1) ).