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Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes.

Kraft JR, Vance RE, Pohl J, Martin AM, Raulet DH, Jensen PE - J. Exp. Med. (2000)

Bottom Line: The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules.The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b).Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

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Binding of Qa-1b tetramers containing substituted Qdm peptides to CD94/NKG2A. (A) Flow cytometry profiles were obtained using CD3−B220− C57BL/10 splenocytes stained with anti-NK1.1 and Qa-1b tetramers, prepared with Qdm or substituted Qdm peptides containing Lys at the indicated position. The Dk leader peptide differs from Qdm, containing a Val instead of Ala at P3. Numbers in the upper right hand corner correspond to percentage of NK1.1+ cells that are tetramer positive. (B) Chinese hamster ovary transfectants expressing CD94 and NKG2A were stained with Qa-1b tetramers (solid line) or streptavidin-allophycocyanin (dotted line).
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Figure 5: Binding of Qa-1b tetramers containing substituted Qdm peptides to CD94/NKG2A. (A) Flow cytometry profiles were obtained using CD3−B220− C57BL/10 splenocytes stained with anti-NK1.1 and Qa-1b tetramers, prepared with Qdm or substituted Qdm peptides containing Lys at the indicated position. The Dk leader peptide differs from Qdm, containing a Val instead of Ala at P3. Numbers in the upper right hand corner correspond to percentage of NK1.1+ cells that are tetramer positive. (B) Chinese hamster ovary transfectants expressing CD94 and NKG2A were stained with Qa-1b tetramers (solid line) or streptavidin-allophycocyanin (dotted line).

Mentions: The above experiments and previous studies 23 clearly indicated that Qa-1b–Qdm complexes can serve as ligands for CD94/NKG2A, but the degree to which the receptor is able to discriminate between Qa-1 complexes containing different peptides remained unknown. This question became particularly relevant in light of the observation that Qa-1 has the potential to bind a relatively diverse repertoire of peptides. This issue was further investigated by generating fluorescent soluble tetrameric Qa-1b complexes bearing Qdm peptides with substitutions at different nonanchor positions. As previously reported 23, tetramers generated from Qa-1b folded with Qdm peptide bind to 40–50% of splenic NK cells (Fig. 5 A). Staining with tetramers containing the Dk leader sequence, which contains Val instead of Ala at P3, was somewhat reduced compared with Qdm, yet a substantial fraction of NK cells clearly binds this reagent. By contrast, little if any staining was observed with tetramers containing substituted Qdm peptides with Lys at P1, P4, P5, or P8. To confirm the specificity of NK cell staining, a parallel experiment was performed with transfectants expressing high levels of CD94/NKG2A. No tetramer staining was observed with control cells that did not express CD94/NKG2A heterodimer 23. Qa-1 tetramers containing Qdm or the Dk leader peptide stained the transfectants uniformly (Fig. 5 B). No staining was observed with peptides containing substitutions at P4, P5, or P8. The transfectants uniformly reacted with tetramer containing the P1-substituted peptide but the mean fluorescence intensity was substantially reduced, suggesting that this substitution may reduce affinity for CD94/NKG2A. The increased staining of transfected cells compared with splenic NK cells with tetramer containing the P1-substituted peptide probably can be attributed to the greater level of expression of CD94/NKG2A on the transfectants.


Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes.

Kraft JR, Vance RE, Pohl J, Martin AM, Raulet DH, Jensen PE - J. Exp. Med. (2000)

Binding of Qa-1b tetramers containing substituted Qdm peptides to CD94/NKG2A. (A) Flow cytometry profiles were obtained using CD3−B220− C57BL/10 splenocytes stained with anti-NK1.1 and Qa-1b tetramers, prepared with Qdm or substituted Qdm peptides containing Lys at the indicated position. The Dk leader peptide differs from Qdm, containing a Val instead of Ala at P3. Numbers in the upper right hand corner correspond to percentage of NK1.1+ cells that are tetramer positive. (B) Chinese hamster ovary transfectants expressing CD94 and NKG2A were stained with Qa-1b tetramers (solid line) or streptavidin-allophycocyanin (dotted line).
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Related In: Results  -  Collection

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Figure 5: Binding of Qa-1b tetramers containing substituted Qdm peptides to CD94/NKG2A. (A) Flow cytometry profiles were obtained using CD3−B220− C57BL/10 splenocytes stained with anti-NK1.1 and Qa-1b tetramers, prepared with Qdm or substituted Qdm peptides containing Lys at the indicated position. The Dk leader peptide differs from Qdm, containing a Val instead of Ala at P3. Numbers in the upper right hand corner correspond to percentage of NK1.1+ cells that are tetramer positive. (B) Chinese hamster ovary transfectants expressing CD94 and NKG2A were stained with Qa-1b tetramers (solid line) or streptavidin-allophycocyanin (dotted line).
Mentions: The above experiments and previous studies 23 clearly indicated that Qa-1b–Qdm complexes can serve as ligands for CD94/NKG2A, but the degree to which the receptor is able to discriminate between Qa-1 complexes containing different peptides remained unknown. This question became particularly relevant in light of the observation that Qa-1 has the potential to bind a relatively diverse repertoire of peptides. This issue was further investigated by generating fluorescent soluble tetrameric Qa-1b complexes bearing Qdm peptides with substitutions at different nonanchor positions. As previously reported 23, tetramers generated from Qa-1b folded with Qdm peptide bind to 40–50% of splenic NK cells (Fig. 5 A). Staining with tetramers containing the Dk leader sequence, which contains Val instead of Ala at P3, was somewhat reduced compared with Qdm, yet a substantial fraction of NK cells clearly binds this reagent. By contrast, little if any staining was observed with tetramers containing substituted Qdm peptides with Lys at P1, P4, P5, or P8. To confirm the specificity of NK cell staining, a parallel experiment was performed with transfectants expressing high levels of CD94/NKG2A. No tetramer staining was observed with control cells that did not express CD94/NKG2A heterodimer 23. Qa-1 tetramers containing Qdm or the Dk leader peptide stained the transfectants uniformly (Fig. 5 B). No staining was observed with peptides containing substitutions at P4, P5, or P8. The transfectants uniformly reacted with tetramer containing the P1-substituted peptide but the mean fluorescence intensity was substantially reduced, suggesting that this substitution may reduce affinity for CD94/NKG2A. The increased staining of transfected cells compared with splenic NK cells with tetramer containing the P1-substituted peptide probably can be attributed to the greater level of expression of CD94/NKG2A on the transfectants.

Bottom Line: The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules.The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b).Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

Show MeSH
Related in: MedlinePlus