Peptide binding motifs of the MHC class I molecules (RT1.Al) of the Lewis rat.

Abstract

Peptide binding to major histocompatibility complex (MHC) class I molecules is governed by allele-specific motifs composed of several conserved anchor positions (reviewed in Rammensee et al. 1995). The peptide binding motifs of the rat MHC class I molecules, RT1.A, have not been well characterized. Thus, except for one preliminary communication (Powis et al. 1993) and a recent report of the RT1.Aa motif (Powis et al. 1996), no other information on peptide specificity of RT1.A molecules has been published. The study of RT1.A motifs is interesting in view of the allelic dimorphism of the rat TAP transporter, which results in different sets of peptides being transported for loading onto RT1.A molecules of different haplotypes (reviewed in Howard 1995). Since the Lewis rat (RT1l) is widely used in the study of transplantation and experimental autoimmune diseases, we sought to characterize a peptide binding motif of RT1.Al using pool and individual sequencing of RT1.Al-bound peptides. In addition, peptide presentation by MHC class I molecules of different cell types has not been studied in detail. In this regard, T cells draw particular attention since MHC class I-restricted interactions between T cells were proposed to play a role in immune regulation (Sun et al. 1988; Zhang et al. 1993; Kuhrober et al. 1994). Therefore, it was of interest to analyze peptides presented by MHC class I molecules on T cells. To this end, we isolated and sequenced peptides naturally bound to MHC class I molecules of the Lewis rat CD4+, TCRαβ+ T cell clone A2b (Holoshitz et al. 1984). During prolonged culture in vitro, a subclone of A2b acquired the ability to continuously grow in the presence of IL-2 without the addition of antigen-presenting cells, while maintaining its antigen specificity and phenotypic markers. The A2b cells were grown in Dulbecco’s modified Eagle medium supplemented with 5% fetal calf serum and rIL-2, pelleted, and detergent-lysed. The lysate was precleared and passed over a column of RT1.A-specific mAb OX18 coupled to Sepharose. Although OX18 was reported to cross-react with non-classical rat class I products (Jameson et al. 1992), these molecules are usually poorly expressed. Indeed, OX18 and another RT1.A-specific mAb, F16-4.4., manifested equally strong staining of A2b cells, suggesting that RT1.A is the major, if not the only, class I protein expressed on these cells. The material bound to the affinity column was eluted with TFA and passed through Centriprep 3 ultrafiltration membranes (Amicon, Beverly, MA). The filtrate was concentrated and separated using reverse-phase HPLC as described (Falk et al. 1994). Dominant single peaks were sequenced individually, whereby the remaining fractions were pooled and sequenced as such. The fractions were sequenced by Edman degradation on a pulsed-liquid protein sequencer 476A with on-line PTH-a.a. analysis (Applied Biosystems Weiterstadt, Germany). The pool sequencing data were interpreted as described (Falk et al. 1991). The results of peptide sequencing are summarized in Table 1. Pool sequencing of RT1.1Al-bound peptides showed two major anchor positions, 3 and 9, occupied by aromatic Phe and Tyr residues. Aliphatic residues such as Leu and Met also contribute to the P9 anchor. An auxiliary anchor was observed at position 2, showing strong signals for small residues such as Ala, Ser, and Val. Minor preferences were detected at other positions, such as small or negatively charged residues at positions 7 and 8. A detailed listing of preferred and other detected amino acids is given in Table 1. In addition, eight individual peptides were sequenced, six of them matching known proteins. All these peptides contain the proposed RT1.Al motif. Although it cannot be formally ruled out that some detected residues could be contributed by non-classical B. Reizis ? F. Mor ? I. R. Cohen ( ) Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100 Israel