Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contai ns a region of sequence similarity, including six identical residues PEVKEK , to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We u sed the human islet cell mAbs MICA3 and MICA4 to identify the Bb epitopes o f GAD65 by screening phage-displayed random peptide libraries. The identifi ed peptide sequences could be mapped to a homology model of the pyridoxal p hosphate (PLP) binding domain of GAD65, For MICAS, a surface loop containin g the sequence PEVKEK and two adjacent exposed helixes were identified in t he PLP binding domain as well as a region of the C terminus of GAD65 that h as previously been identified as critical for MICA3 binding. To confirm tha t the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis, This reduced binding of MICA3 by 70% , Peptide sequences selected using MICA4 were rich in basic or hydroxyl-con taining amino acids, and the surface of the CAD65 PLP-binding domain surrou nding Lys(358), which is known to be critical for MICA4 binding, was likewi se rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and muta genesis and have provided compelling evidence for the involvement of the PE VKEK loop in the MICA3 epitope.