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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 residues P2 and P3 determine Ly49C recognition of H-2Kb.(A) Expression of H-2Kb incubated with the indicated peptides. (B) Percent cytotoxicity of RMA-S targets incubated with the indicated peptides incubated with RNK.49W/C effectors. (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 < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.
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pone.0131308.g005: Peptide residues P2 and P3 determine Ly49C recognition of H-2Kb.(A) Expression of H-2Kb incubated with the indicated peptides. (B) Percent cytotoxicity of RMA-S targets incubated with the indicated peptides incubated with RNK.49W/C effectors. (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 < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.

Mentions: We further investigated the role of peptide residues in Ly49W/C recognition of H-2Kb by comparing variants of a synthetic peptide, AAYAYAAL. Like RGYVYQGL, AAYAYAAL is not supportive of H-2Kb interaction with Ly49W/C. We recapitulated the identity of SIINFEKL residues that we and others have found to contribute to Ly49C recognition into the AAYAYAAL peptide to test gain of recognition. Peptides used in this assay include AIIAFAKL, AIIAFAAL, and AIIAYAAL, that bind H-2Kb at similar levels, demonstrated by RMA/S stabilization assays that were performed in parallel to cytotoxicity assays (Fig 5A). Interestingly, the peptide AIIAFAKL is fully supportive of Ly49W/C recognition, as is AIIAFAAL and AIIAYAAL, suggesting a pivotal role for residues that dock into the adjacent B-pocket and D-pocket of H-2Kb which might be a site important to allow for H-2Kb and Ly49W/C interaction (Fig 5B and 5C). To pinpoint individual effects of P2 and P3 in Ly49W/C recognition of H-2Kb, we tested P2 and P3 Ala substitutions in the AIIAYAAL peptide. However, peptides AAIAYAAL and AIAAYAAL did not stabilize H-2Kb on RMA-S cells for the duration of the cytotoxicity assays (S1 Fig). Nevertheless, our data indicate that specific amino acid identities at both P3 and P2 peptide residues are required for optimal H-2Kb recognition by Ly49W/C.


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

Marquez EA, Kane KP - PLoS ONE (2015)

Peptide residues P2 and P3 determine Ly49C recognition of H-2Kb.(A) Expression of H-2Kb incubated with the indicated peptides. (B) Percent cytotoxicity of RMA-S targets incubated with the indicated peptides incubated with RNK.49W/C effectors. (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 < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493100&req=5

pone.0131308.g005: Peptide residues P2 and P3 determine Ly49C recognition of H-2Kb.(A) Expression of H-2Kb incubated with the indicated peptides. (B) Percent cytotoxicity of RMA-S targets incubated with the indicated peptides incubated with RNK.49W/C effectors. (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 < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.
Mentions: We further investigated the role of peptide residues in Ly49W/C recognition of H-2Kb by comparing variants of a synthetic peptide, AAYAYAAL. Like RGYVYQGL, AAYAYAAL is not supportive of H-2Kb interaction with Ly49W/C. We recapitulated the identity of SIINFEKL residues that we and others have found to contribute to Ly49C recognition into the AAYAYAAL peptide to test gain of recognition. Peptides used in this assay include AIIAFAKL, AIIAFAAL, and AIIAYAAL, that bind H-2Kb at similar levels, demonstrated by RMA/S stabilization assays that were performed in parallel to cytotoxicity assays (Fig 5A). Interestingly, the peptide AIIAFAKL is fully supportive of Ly49W/C recognition, as is AIIAFAAL and AIIAYAAL, suggesting a pivotal role for residues that dock into the adjacent B-pocket and D-pocket of H-2Kb which might be a site important to allow for H-2Kb and Ly49W/C interaction (Fig 5B and 5C). To pinpoint individual effects of P2 and P3 in Ly49W/C recognition of H-2Kb, we tested P2 and P3 Ala substitutions in the AIIAYAAL peptide. However, peptides AAIAYAAL and AIAAYAAL did not stabilize H-2Kb on RMA-S cells for the duration of the cytotoxicity assays (S1 Fig). Nevertheless, our data indicate that specific amino acid identities at both P3 and P2 peptide residues are required for optimal H-2Kb recognition by Ly49W/C.

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