0014-2980/98/0404-1272$17.50 + .50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998 Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries James Stevens 1 , Karl-Heinz Wiesmüller 2 , Peter Walden 3 and Etienne Joly 1 1 Department of Immunology, The Babraham Institute, Cambridge, GB 2 Naturwissenschaftliches und Medizinisches Institut, Universität Tübingen, Reutlingen, Germany 3 Dermatologische Klinik, Charit ´ e, Humboldt-Universität, Berlin, Germany MHC class I molecules bind short peptides for presentation to CD8 + T cells. The determina- tion of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1 c , which carries unusual key residues at both ends of the groove, and RT1.A a which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using syn- thetic random peptide libraries of different lengths (7–15 amino acids) and successful stabili- zation was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-K b , 8–13-mer and H2-D b , 9–15- mer peptides). The rat MHC class Ia molecule, RT1-A a , revealed a preference for 9–15-mer peptides, whereas RT1-A1 c showed a more stringent preference for 9–12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides. Key words: MHC class I/Random peptide library / Flow cytometry analysis / RT1-A / Peptide sta- bilization Received 12/12/97 Accepted 26/1/98 [I 17841] 1 Introduction The class I molecules of the major histocompatibility complex (MHC) are heterodimers composed of a vari- able membrane-spanning heavy chain non-covalently associated with a light chain, 2-microglobulin ( 2m). After synthesis and folding in the endoplasmic reticulum (ER) they are loaded with peptides, which they then carry to the cell surface for presentation to CD8 + T cells. The peptides found bound to MHC class I molecules at the surface of cells have been generated by mechanisms which are potentially selective, e.g. proteolysis, trans- membrane transport and chaperone binding. One of the main sites for peptide generation is the cytoplasm where soluble proteases and the proteasome, a large multicat- alytic protease complex, are present. The proteasome was found to be implicated in antigenic peptide genera- tion by Rock et al. [1] and two proteasome subunits, LMP2 and LMP7, encoded in the MHC region [2], have been found to influence the scope of peptides presented by MHC class I molecules. The proteasome, however, is possibly not the only source of peptide for MHC mole- cules [3]. Once peptides are made, they are transported into the ER via the heterodimeric transporter associated with antigen presentation (TAP) [4, 5] for association to MHC class I molecules. Allelic variants of TAP have been described for human [6–9], mouse [10] and rat [11]. Whereas no func- tional polymorphisms have yet been found in mouse or human TAP alleles [12–14], the two rat TAP2 alleles were found to differ by as much as 25 amino acids, and this was subsequently shown to affect the supply of peptides to MHC class I molecules [11, 12, 15]. The TAP-A form of the transporter (TAP1/TAP2-A) can efficiently transport peptides with many different amino acids present at the C terminus, whereas the TAP-B transporter (TAP1/TAP2-B) can only efficiently transport those with hydrophobic and aromatic residues at the terminal position [12, 16]. In addi- tion, the TAP-B form of the transporter has been reported to be able to accept peptides longer than the usual 8–11 amino acids [17] although this is disputed by the data of Koopmann et al. [18]. 1272 J. Stevens et al. Eur. J. Immunol. 1998. 28: 1272–1279