Conserved cysteine residues within the E3/19K protein of adenovirus type 2 are essential for binding to major histocompatibility complex antigens.

AUTOR(ES)

Sester, M

RESUMO

The E3/19K protein of human adenovirus type 2 is a resident transmembrane glycoprotein of the endoplasmic reticulum. Its capacity to associate with class I histocompatibility (MHC) antigens abrogates cell surface expression and the antigen presentation function of MHC antigens. At present, it is unclear exactly which structure of the E3/19K protein mediates binding to MHC molecules. Apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, E3/19K molecules from different adenovirus subgroups (B and C) share little homology. Remarkably, the majority of cysteines are conserved. In this report, we examined the importance of cysteine residues for the structure and function of E3/19K. We show that E3/19K contains intramolecular disulfide bonds. By using site-directed mutagenesis, individual cysteines were replaced by serines and mutant proteins were stably expressed in 293 cells. On the basis of the differential binding of monoclonal antibody Tw1.3 and cyanogen bromide cleavage experiments, a structural model of E3/19K is proposed, in which Cys-11 and Cys-28 as well as Cys-22 and Cys-83 are linked by disulfide bonds. Both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human MHC antigens. This was demonstrated by three criteria: loss of E3/19K coprecipitation, lack of transport inhibition, and normal cell surface expression of MHC molecules. Mutation of the three other cysteines had no effect. This indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the E3/19K molecule, namely, to bind and to inhibit transport of MHC antigens.

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