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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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Structural characterization of the tumor epitope. (A) CAD mass spectrum recorded from the (M+H)+1 ions (m/z 854) of the tumor antigen.  (B) CAD mass spectrum recorded on (M+H)+1 ions (m/z 767) from the tumor antigen after a single round of Edman degradation. The ions observed in  each spectra are underlined. (C) Results of coelution experiments in which synthetic peptides SNFVFAGL or SNFVFAGI were added to the biologically  active subfraction 37-19 containing the tumor antigen. (D) synthetic peptides SNFVFAGI and VTFVFAGX (X = L or I) were loaded onto RMA-S cells  in the indicated concentrations, and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. SNFVFAGI is specifically recognized by the anti-A  CTL clone.
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Figure 6: Structural characterization of the tumor epitope. (A) CAD mass spectrum recorded from the (M+H)+1 ions (m/z 854) of the tumor antigen. (B) CAD mass spectrum recorded on (M+H)+1 ions (m/z 767) from the tumor antigen after a single round of Edman degradation. The ions observed in each spectra are underlined. (C) Results of coelution experiments in which synthetic peptides SNFVFAGL or SNFVFAGI were added to the biologically active subfraction 37-19 containing the tumor antigen. (D) synthetic peptides SNFVFAGI and VTFVFAGX (X = L or I) were loaded onto RMA-S cells in the indicated concentrations, and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. SNFVFAGI is specifically recognized by the anti-A CTL clone.

Mentions: To determine the sequence of the tumor antigen an aliquot from the remaining 40% of subfraction 36-19 was loaded onto a C18 microcapillary column (75 μm i.d. × 12 cm) and eluted with a 12-min gradient (0–80% acetonitrile in 0.1 M acetic acid) directly into a triple quadruple mass spectrometer (Finnigan MAT, TSQ7000) essentially as described (34, 35). This instrument is equipped with an electrospray ionization source that was operated with a coaxial sheath (70% MeOH/H2O containing 0.12% acetic acid) flowing at 1.5 μl/min. A negative potential of 4.6 kV was applied to the heated capillary. Quadrupole one was set to pass a 2 mass unit window centered on 854, the m/z value corresponding to the (M + H)+1 ions of the tumor antigen. Ions of this mass were transmitted to quadrupole 2 where they suffered collision-activated dissociation (CAD). The resulting fragments were mass analyzed in quadrupole 3 to produce the CAD mass spectrum shown in Fig. 6.


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)

Structural characterization of the tumor epitope. (A) CAD mass spectrum recorded from the (M+H)+1 ions (m/z 854) of the tumor antigen.  (B) CAD mass spectrum recorded on (M+H)+1 ions (m/z 767) from the tumor antigen after a single round of Edman degradation. The ions observed in  each spectra are underlined. (C) Results of coelution experiments in which synthetic peptides SNFVFAGL or SNFVFAGI were added to the biologically  active subfraction 37-19 containing the tumor antigen. (D) synthetic peptides SNFVFAGI and VTFVFAGX (X = L or I) were loaded onto RMA-S cells  in the indicated concentrations, and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. SNFVFAGI is specifically recognized by the anti-A  CTL clone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196148&req=5

Figure 6: Structural characterization of the tumor epitope. (A) CAD mass spectrum recorded from the (M+H)+1 ions (m/z 854) of the tumor antigen. (B) CAD mass spectrum recorded on (M+H)+1 ions (m/z 767) from the tumor antigen after a single round of Edman degradation. The ions observed in each spectra are underlined. (C) Results of coelution experiments in which synthetic peptides SNFVFAGL or SNFVFAGI were added to the biologically active subfraction 37-19 containing the tumor antigen. (D) synthetic peptides SNFVFAGI and VTFVFAGX (X = L or I) were loaded onto RMA-S cells in the indicated concentrations, and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. SNFVFAGI is specifically recognized by the anti-A CTL clone.
Mentions: To determine the sequence of the tumor antigen an aliquot from the remaining 40% of subfraction 36-19 was loaded onto a C18 microcapillary column (75 μm i.d. × 12 cm) and eluted with a 12-min gradient (0–80% acetonitrile in 0.1 M acetic acid) directly into a triple quadruple mass spectrometer (Finnigan MAT, TSQ7000) essentially as described (34, 35). This instrument is equipped with an electrospray ionization source that was operated with a coaxial sheath (70% MeOH/H2O containing 0.12% acetic acid) flowing at 1.5 μl/min. A negative potential of 4.6 kV was applied to the heated capillary. Quadrupole one was set to pass a 2 mass unit window centered on 854, the m/z value corresponding to the (M + H)+1 ions of the tumor antigen. Ions of this mass were transmitted to quadrupole 2 where they suffered collision-activated dissociation (CAD). The resulting fragments were mass analyzed in quadrupole 3 to produce the CAD mass spectrum shown in Fig. 6.

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