The Journal of Experimental Medicine
VeriKine-HS Human IFN-Beta
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© The Rockefeller University Press, 0022-1007/1997/3/1013/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 6, March 17, 1997 1013-1022

Articles

A Peptide-binding Motif for I-Ag7, the Class II Major Histocompatibility Complex (MHC) Molecule of NOD and Biozzi AB/H Mice

Leonard C. Harrison*, Margo C. Honeyman*, Sylvie Trembleau{ddagger}, Silvia Gregori{ddagger}, Fabio Gallazzi{ddagger}, Petra Augstein*, Vladimir Brusic*, Juergen Hammer{ddagger}, and Luciano Adorini{ddagger}

From * the Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville 3050, Australia; and {ddagger} Roche Milano Ricerche, 1-20132, Milano, Italy

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Address correspondence to L.C. Harrison, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville, 3050, Australia.

L.C. Harrison and M.C. Honeyman were supported by the National Health and Medical Research Council of Australia and a Diabetes Interdisciplinary Research Program grant from the Juvenile Diabetes Foundation International. P. Augstein was supported by Deutscher Akademischer Austauschdienst, Bonn. Secretarial assistance was provided by Margaret Thompson.

1 Abbreviations used in this paper: CR-EAE, chronic relapsing experimental allergic encephalomyelitis; DOC, sodium deoxycholate; EAE, experimental allergic encephalomyelitis; GAD, glutamic acid decarboxylase; HEL, hen egg white lysozyme; IDDM, insulin-dependent diabetes mellitus; MOG, myelin oligodendrocyte glycoprotein; MSA, mouse serum albumin; NOD, nonobese diabetic; OGP, octyl-β-D-glucopyranoside; p, position; PEG, polyethylene glycol; PLP, proteolypid protein; RT, room temperature.


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