Immunogenetics (1995) 42:302-303 © Springer-Verlag 1995 PEPTIDE MOTIF REGISTER Nagendra R. Hegde • Shirley A. Ellis • Ruth M. Gaddum Clive A. Tregaskes • Gautam Sarath Subramaniam Srikumaran Peptide motif of the cattle MHC class I antigen BoLA-A11 Received: 2 May 1995 MHC class I molecules present self-peptides as well as peptides derived from intracellular pathogens to receptors on cytotoxic T lymphocytes (CTLs). The bound peptides are usually 8-10 amino acids long, and generally have one, two, or more chemically related amino acid residue(s) recurring at two or more positions. These anchor residues define the peptide binding characteristics of an allelic product (for a review see Rammensee et al. 1993). Co- dominant expression of MHC alleles has made it necessary either to use allele-specific monoclonal antibodies (mAbs) or to use single allelic transfectants for the characterization of allele-specific peptide motifs. Data from several bovine lymphocyte antigen (BoLA) workshops have demonstrated the existence of more than 50 serologically defined MHC class I specificities (Davies et al. 1994). It is apparent that these specificities may encompass more than one gene product, and it is at present unclear how many of the three (or more) class I genes are transcribed (Davies and co-workers, unpublished data). In this study, we report the peptide motif for a BoLA-All gene product. Mouse fibroblasts (L cells) were transfected with sheared genomic DNA from a heterozygous animal which typed serologically as All/A14. Transfectants were screened initially for cattle MHC class I expression using the mAb IL-A88 (Toye et al. 1990), which recognizes a monomorphic determinant on cattle MHC class I heavy chains, and subsequently for the All specificity, by allo- specific sera. A gene encoding a product recognized as BoLA-A11 has been recently cloned, sequenced, and trans- fected (Sawhney et al. 1995). The transfected cell line used N_ R. Hegde • S. Srikumaran (~) Dept. of Veterinaryand Biomedical Sciences,University of Nebraska- Lincoln, Lincoln, NE 68583-0905, USA G. Sarath Dept. of Biochemistry and Protein Core Facility, University of Nebraska-Lincoln, Lincoln, NE 68583-0718, USA S. A. Ellis • R. M. Gaddum • C. A. Tregaskes Institute for Animal Health, Compton Laboratory,Compton, Nr. Newbury, RG20 7NN, UK in our study is indistinguishable by serology and 1D IEF analysis from the cell line described by Sawhney and co- workers (data not shown). Transfectants were grown (to 1 ×10 ~0) and lysed in the presence of 0.5% NP-40. Immunoaffinity columns were made by covalently cross-linking Protein A (Pharmacia, Piscataway, NJ), using dimethylpimelimidate (Sigma, St. Louis, MO), with the mAb [L-A88, or an isotype-matched control mAb (specific for the envelope glycoprotein gp53 of bovine viral diarrhoea virus). The lysate was passed first over the control mAb column, then over the anti-class I mAb column. The cattle class I molecules were eluted, and dissociated from bound peptides by acid teatment (0.1% trifluoroacetic acid). The peptide pool was separated from the MHC class I heavy chain and ~2-microglobulin by using a 3000 Mr cut-off membrane filter (Amicon, Beverly, MA). The peptide pool was analyzed by sequential Edman degradation (Millipore 6600 Protein Sequencer; Millipore, Predford, MA) for 12 cycles according to the manufac- turer's protocol, and the results were interpreted as de- scribed in Falk and co-workers (1991). Table 1 shows the positional characteristics for peptides bound to the BoLA- All allelic product, obtained from three independent ex- periments and five independent sequence analyses. The majority of the peptides that occupied the binding groove of BoLA-All were nonamers. There was clear evidence that position 2 was occupied by Pro, although a small amount of Gln was observed in some experiments. The carboxy-terminal amino acid anchor was Ile/Val with a small proportion of Leu and Tyr. Ala and Pro appeared to be the preferred residues for P3 and P5, respectively, and these positions may serve as auxiliary anchors. Pronounced heterogeneity occurred at P3, while a lesser degree of heterogeneity was seen at other positions of the peptide. Tyr produced a significant signal at P10, while signals at Pll and P12 were not obtained in all of the experiments (see Table 1). The possibility of a few decamer peptides associated with B oLA-A 11 cannot be ruled out, particularly since Pro at one or more dominant and/or auxiliary anchor positions can kink the peptide, so that the C/F pocket, which has been implicated in accomodating P9 (Saper et al.