Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain

Membrane-bound immunoglobulins have, in addition to the transmembrane and c ytoplasmic portions, an extracellular membrane-proximal domain (EMPD), abse nt in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, a nd epsilon contain cysteines whose role has so far not been elucidated. Usi ng a genetic strategy, we investigated the ability of these cysteines to fo rm disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were c onstructed and expressed on the surface of a B-cell line. The resulting mem brane proteins contain a single chain fragment of variable regions (scFv) l inked to the dimerizing domain from the immunoglobulin heavy chains (CH3 fo r alpha and gamma or CH4 for epsilon isotypes), followed by the correspondi ng EMPD and the transmembrane and cytoplasmic domains. The two functional m embrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha 1L an d gamma1 EMPD and the short version of epsilon EMPD form an interchain disu lfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon -long EMPD contains four cysteines; two a re involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy ch ain mutant, in which Cys(121) and Cyd(209) within domain CH2 (involved in i nterchain bridges) were mutated to alanines, confirmed that, within the com plete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, w e unveil evidence for additional covalent stabilization of membrane-bound i mmunoglobulins.