Abstract
Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1(c) but not other rat MHC class Ia or Ib molecules. RT1-A1(c) preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1(c) could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1(c) and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1(c) primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1(c)-bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1(c) with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1(c) bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1(c) bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1(c) is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2.
Highlights
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RT1-A1c preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC class I (MHC-I) molecule
The rat MHC class Ia molecule RT1-A1c can be classified into the HLA-B7 supertype based on its preference for binding nonamer peptides with proline as the second residue from the N terminus at the P2 anchor position (Fig. 1A) [12]
Summary
To test the importance of bound peptide P2 anchor residue identity in Ly49i2 recognition of RT1-A1c, we synthesized peptides for binding to RT1-A1c that shared the “ideal RT1-A1c binding peptide” sequence, with the exception that they each differed in the amino acid Pro, Gln, or Val at the P2 anchor position: NPRKVTAYL (P2P), NQRKVTAYL (P2Q), and NVRKVTAYL (P2V) (Fig. 1B). Using a rat NK leukemia cell line, RNK-16, stably transfected to express Ly49i2, our laboratory previously showed that recognition of RT1A1c is dependent on the composition of MHC-I residues of the Bpocket, which determines the P2 anchor residue accepted and in turn defines the MHC-I supertype [13].
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