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Identities of P2 and P3 Residues of H-2Kb-Bound Peptides Determine Mouse Ly49C Recognition.

Marquez EA, Kane KP - PLoS ONE (2015)

Bottom Line: Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others.Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity.If both are aliphatic residues, this is supportive.

View Article: PubMed Central - PubMed

Affiliation: Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others. Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity. If both are aliphatic residues, this is supportive. Whereas, small amino acids at P2 and aromatic amino acids at the P3 auxiliary anchor residue are detrimental to Ly49C recognition. These results resemble those with a rat Ly49 where the identity of a peptide anchor residue determines recognition, suggesting that dependence on specific peptide residues buried in the MHC-I peptide-binding groove may be fundamental to Ly49 peptide selectivity and recognition.

No MeSH data available.


Related in: MedlinePlus

Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition.(A) Expression of H-2Kb on RMA/S cells co-incubated with the listed peptides (B) Cytotoxic assay using RNK.49W/C effector cells and RMA-S targets incubated with SIINFEKL or the indicated SIINFEKL variants (SIVNFEKL and SIYNFEKL) and RGYVYQGL (C) Statistically significant changes in Ly49W/C recognition of H-2Kb, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (*p < 0.05, **p < 0.005). Results plotted are the mean of three independent experiments with error bars indicating SD.
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pone.0131308.g003: Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition.(A) Expression of H-2Kb on RMA/S cells co-incubated with the listed peptides (B) Cytotoxic assay using RNK.49W/C effector cells and RMA-S targets incubated with SIINFEKL or the indicated SIINFEKL variants (SIVNFEKL and SIYNFEKL) and RGYVYQGL (C) Statistically significant changes in Ly49W/C recognition of H-2Kb, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (*p < 0.05, **p < 0.005). Results plotted are the mean of three independent experiments with error bars indicating SD.

Mentions: In order to corroborate auxiliary anchor residue P3 directed recognition, we considered an Ala substituted SIINFEKL variant at P3 to directly examine Ly49W/C recognition. However, the SIINFEKL analog bearing Ala at P3 does not stably bind H-2Kb compared to the unchanged SIINFEKL peptide, as previously reported [33]. Therefore, we tested recognition by substituting the identity of residues at P3 between RGYVYQGL peptide, that does not confer Ly49C and H-2Kb interaction, and SIINFEKL, a peptide supportive of Ly49C interaction with H-2Kb, creating the SIYNFEKL peptide. In addition, we tested a SIINFEKL variant with Val at P3, similar to Ile, that can allow us to examine preferred residue chemistry at P3 for Ly49W/C and H-2Kb association. In parallel to killing assays, we also carried out RMA/S stabilization assays with peptides SIVNFEKL and SIYNFEKL, as well as with control peptides, where all peptides yielded H-2Kb expression at similar levels (Fig 3A). Substituting Val for Ile at P3, SIVNFEKL, made no significant difference to H-2Kb recognition, as observed in cytotoxicity experiments; however, exchange of a Tyr for Ile, SIYNFEKL, partially reduced H-2Kb recognition as compared to the RGYVYQGL peptide or no peptide (Fig 3B and 3C). These results indicated that aliphatic residues Val or Ile at P3 are fully supportive of, while the aromatic and polar Tyr is detrimental to, Ly49W/C interaction with H-2Kb. The substitution of Tyr for Ile at P3 in SIINFEKL did not completely disrupt recognition, indicating that additional residue(s) may contribute to Ly49C and H-2Kb interaction. Nevertheless, the identity of the P3 auxiliary anchor residue of bound peptide is an important factor in H-2Kb recognition.


Identities of P2 and P3 Residues of H-2Kb-Bound Peptides Determine Mouse Ly49C Recognition.

Marquez EA, Kane KP - PLoS ONE (2015)

Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition.(A) Expression of H-2Kb on RMA/S cells co-incubated with the listed peptides (B) Cytotoxic assay using RNK.49W/C effector cells and RMA-S targets incubated with SIINFEKL or the indicated SIINFEKL variants (SIVNFEKL and SIYNFEKL) and RGYVYQGL (C) Statistically significant changes in Ly49W/C recognition of H-2Kb, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (*p < 0.05, **p < 0.005). Results plotted are the mean of three independent experiments with error bars indicating SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493100&req=5

pone.0131308.g003: Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition.(A) Expression of H-2Kb on RMA/S cells co-incubated with the listed peptides (B) Cytotoxic assay using RNK.49W/C effector cells and RMA-S targets incubated with SIINFEKL or the indicated SIINFEKL variants (SIVNFEKL and SIYNFEKL) and RGYVYQGL (C) Statistically significant changes in Ly49W/C recognition of H-2Kb, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (*p < 0.05, **p < 0.005). Results plotted are the mean of three independent experiments with error bars indicating SD.
Mentions: In order to corroborate auxiliary anchor residue P3 directed recognition, we considered an Ala substituted SIINFEKL variant at P3 to directly examine Ly49W/C recognition. However, the SIINFEKL analog bearing Ala at P3 does not stably bind H-2Kb compared to the unchanged SIINFEKL peptide, as previously reported [33]. Therefore, we tested recognition by substituting the identity of residues at P3 between RGYVYQGL peptide, that does not confer Ly49C and H-2Kb interaction, and SIINFEKL, a peptide supportive of Ly49C interaction with H-2Kb, creating the SIYNFEKL peptide. In addition, we tested a SIINFEKL variant with Val at P3, similar to Ile, that can allow us to examine preferred residue chemistry at P3 for Ly49W/C and H-2Kb association. In parallel to killing assays, we also carried out RMA/S stabilization assays with peptides SIVNFEKL and SIYNFEKL, as well as with control peptides, where all peptides yielded H-2Kb expression at similar levels (Fig 3A). Substituting Val for Ile at P3, SIVNFEKL, made no significant difference to H-2Kb recognition, as observed in cytotoxicity experiments; however, exchange of a Tyr for Ile, SIYNFEKL, partially reduced H-2Kb recognition as compared to the RGYVYQGL peptide or no peptide (Fig 3B and 3C). These results indicated that aliphatic residues Val or Ile at P3 are fully supportive of, while the aromatic and polar Tyr is detrimental to, Ly49W/C interaction with H-2Kb. The substitution of Tyr for Ile at P3 in SIINFEKL did not completely disrupt recognition, indicating that additional residue(s) may contribute to Ly49C and H-2Kb interaction. Nevertheless, the identity of the P3 auxiliary anchor residue of bound peptide is an important factor in H-2Kb recognition.

Bottom Line: Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others.Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity.If both are aliphatic residues, this is supportive.

View Article: PubMed Central - PubMed

Affiliation: Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others. Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity. If both are aliphatic residues, this is supportive. Whereas, small amino acids at P2 and aromatic amino acids at the P3 auxiliary anchor residue are detrimental to Ly49C recognition. These results resemble those with a rat Ly49 where the identity of a peptide anchor residue determines recognition, suggesting that dependence on specific peptide residues buried in the MHC-I peptide-binding groove may be fundamental to Ly49 peptide selectivity and recognition.

No MeSH data available.


Related in: MedlinePlus