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The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation.This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

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The mutant p68 peptide sensitizes RMA-S cells for lysis by the  anti-A CTL clone, and stabilizes MHC class I on the cell surface, but the  corresponding wild-type peptide does not. The mutant peptide SNFVFAGI and the wild-type peptide SNFVSAGI were loaded onto RMA-S  cells in the indicated amounts. (A) the loaded cells were tested for lysis by  the anti-A CTL clone in a 51Cr-release assay at an E/T ratio of 2:1. (B)  loaded cells were analyzed for cell surface expression of H-2Kb using fluorescence activated cell sorter analysis, by indirect immunofluorescence with  the monoclonal anti-H2-Kb antibody Y-3 and a polyclonal goat anti– mouse IgG conjugated to fluorescein isothiocyanate as a second step. Percent stabilization of H-2Kb was calculated by subtracting the amount of  H-2Kb present on RMA-S cells shifted to 37°C without added peptide,  from the amount of H-2Kb present on RMA-S cells shifted to 37°C in  the presence of peptide, and dividing by the amount of H-2Kb present on  RMA-S cells which had been kept at room temperature throughout the  experiment.
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Figure 8: The mutant p68 peptide sensitizes RMA-S cells for lysis by the anti-A CTL clone, and stabilizes MHC class I on the cell surface, but the corresponding wild-type peptide does not. The mutant peptide SNFVFAGI and the wild-type peptide SNFVSAGI were loaded onto RMA-S cells in the indicated amounts. (A) the loaded cells were tested for lysis by the anti-A CTL clone in a 51Cr-release assay at an E/T ratio of 2:1. (B) loaded cells were analyzed for cell surface expression of H-2Kb using fluorescence activated cell sorter analysis, by indirect immunofluorescence with the monoclonal anti-H2-Kb antibody Y-3 and a polyclonal goat anti– mouse IgG conjugated to fluorescein isothiocyanate as a second step. Percent stabilization of H-2Kb was calculated by subtracting the amount of H-2Kb present on RMA-S cells shifted to 37°C without added peptide, from the amount of H-2Kb present on RMA-S cells shifted to 37°C in the presence of peptide, and dividing by the amount of H-2Kb present on RMA-S cells which had been kept at room temperature throughout the experiment.

Mentions: The H-2Kb-binding motif (40) predicts that, first, the anchor residue for binding to the molecule is at position five of the peptide, and is an aromatic residue, either phenylalanine or tyrosine, and second, that position eight of the peptide is either a leucine or isoleucine. This sequence motif predicts that the normal homologue of the A antigen peptide, which has serine at position five, would not bind to H-2Kb. Consistent with this prediction, we found that the wild-type peptide SNFVSAGI, in contrast to the mutant peptide SNFVFAGI, neither sensitized RMA-S cells for lysis by the anti-A CTL clone (Fig. 8 A) nor bound effectively to H2-Kb as measured by stabilization of H2-Kb on the surface of RMA-S cells (Fig. 8 B).


The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

The mutant p68 peptide sensitizes RMA-S cells for lysis by the  anti-A CTL clone, and stabilizes MHC class I on the cell surface, but the  corresponding wild-type peptide does not. The mutant peptide SNFVFAGI and the wild-type peptide SNFVSAGI were loaded onto RMA-S  cells in the indicated amounts. (A) the loaded cells were tested for lysis by  the anti-A CTL clone in a 51Cr-release assay at an E/T ratio of 2:1. (B)  loaded cells were analyzed for cell surface expression of H-2Kb using fluorescence activated cell sorter analysis, by indirect immunofluorescence with  the monoclonal anti-H2-Kb antibody Y-3 and a polyclonal goat anti– mouse IgG conjugated to fluorescein isothiocyanate as a second step. Percent stabilization of H-2Kb was calculated by subtracting the amount of  H-2Kb present on RMA-S cells shifted to 37°C without added peptide,  from the amount of H-2Kb present on RMA-S cells shifted to 37°C in  the presence of peptide, and dividing by the amount of H-2Kb present on  RMA-S cells which had been kept at room temperature throughout the  experiment.
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Related In: Results  -  Collection

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Figure 8: The mutant p68 peptide sensitizes RMA-S cells for lysis by the anti-A CTL clone, and stabilizes MHC class I on the cell surface, but the corresponding wild-type peptide does not. The mutant peptide SNFVFAGI and the wild-type peptide SNFVSAGI were loaded onto RMA-S cells in the indicated amounts. (A) the loaded cells were tested for lysis by the anti-A CTL clone in a 51Cr-release assay at an E/T ratio of 2:1. (B) loaded cells were analyzed for cell surface expression of H-2Kb using fluorescence activated cell sorter analysis, by indirect immunofluorescence with the monoclonal anti-H2-Kb antibody Y-3 and a polyclonal goat anti– mouse IgG conjugated to fluorescein isothiocyanate as a second step. Percent stabilization of H-2Kb was calculated by subtracting the amount of H-2Kb present on RMA-S cells shifted to 37°C without added peptide, from the amount of H-2Kb present on RMA-S cells shifted to 37°C in the presence of peptide, and dividing by the amount of H-2Kb present on RMA-S cells which had been kept at room temperature throughout the experiment.
Mentions: The H-2Kb-binding motif (40) predicts that, first, the anchor residue for binding to the molecule is at position five of the peptide, and is an aromatic residue, either phenylalanine or tyrosine, and second, that position eight of the peptide is either a leucine or isoleucine. This sequence motif predicts that the normal homologue of the A antigen peptide, which has serine at position five, would not bind to H-2Kb. Consistent with this prediction, we found that the wild-type peptide SNFVSAGI, in contrast to the mutant peptide SNFVFAGI, neither sensitized RMA-S cells for lysis by the anti-A CTL clone (Fig. 8 A) nor bound effectively to H2-Kb as measured by stabilization of H2-Kb on the surface of RMA-S cells (Fig. 8 B).

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation.This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

Show MeSH
Related in: MedlinePlus