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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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Sequential HPLC separation of sensitizing peptides eluted from  the H2-Kb molecule of 8101-RE cells. A shows the results of the firstdimension microbore HPLC separation of peptides eluted from 5 × 1010  8101-RE cells, using HFBA as the ionic modifier. A portion (0.3%) of  each fraction was loaded onto RMA-S cells and tested for lysis by the  anti-A CTL clone in a 51Cr-release assay. Sensitizing fractions 36 and 37  were subfractionated on the same microbore column using TFA as the  ionic modifier (B and C). RMA-S cells were loaded with 1.5% of each  subfraction and tested for lysis by the anti-A CTL clone. The E/T ratio  was 5:1. Peptide loaded RMA-S cell with CTL (solid bars) or without  CTL (hatched bars) as toxicity controls are shown in A. Toxicity controls  were not done in B and C.
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Figure 4: Sequential HPLC separation of sensitizing peptides eluted from the H2-Kb molecule of 8101-RE cells. A shows the results of the firstdimension microbore HPLC separation of peptides eluted from 5 × 1010 8101-RE cells, using HFBA as the ionic modifier. A portion (0.3%) of each fraction was loaded onto RMA-S cells and tested for lysis by the anti-A CTL clone in a 51Cr-release assay. Sensitizing fractions 36 and 37 were subfractionated on the same microbore column using TFA as the ionic modifier (B and C). RMA-S cells were loaded with 1.5% of each subfraction and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. Peptide loaded RMA-S cell with CTL (solid bars) or without CTL (hatched bars) as toxicity controls are shown in A. Toxicity controls were not done in B and C.

Mentions: To isolate the naturally processed A antigen peptide, we immunoprecipitated the H-2Kb molecules from 5 × 1010 8101-RE tumor cells. Associated peptides were eluted from the MHC class I molecule with acid, and separated from high molecular mass proteins by filtration through a 5-kD molecular mass cutoff membrane. The unfractionated peptide extract sensitized RMA-S cells for lysis by the anti-A CTL clone (data not shown). The peptide extract was concentrated and then fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) using heptafluorobutyric acid (HFBA) as the ionic modifier. Aliquots from individual fractions were tested. Only two fractions, 36 and 37, sensitized RMA-S cells for lysis by an anti-A CTL clone (Fig. 4 A). Active fractions 36 and 37 were individually rechromatographed over the same HPLC column, using a shallower gradient and TFA as the ionic modifier. A peptide in subfraction 36-19, 36-20 (Fig. 4 B), and 37-19 (Fig. 4 C) sensitized RMA-S cells for lysis by the anti-A CTL clone. However, each subfraction contained more than 100 peptides, too many to sequence with the available material. Therefore, to identify the mass of the A antigen in these mixtures, 60% of each HPLC subfraction was analyzed separately (the remaining 40% was set aside for further analysis) with an on-line microcapillary column effluent splitter, as described previously (33). Four-fifths of the effluent is directed into the mass spectrometer for analysis, while the other one-fifth is simultaneously deposited into a 96-well plate for analysis of sensitizing activity. Since both pieces of data are acquired as a function of time, the ion abundance corresponding to a particular peptide can be correlated with the biological activity. The microcapillary split of subfraction 36-19 and 37-19 yielded activity (Fig. 5, A and B, respectively) but no sensitizing activity was observed from the peptides in subfraction 36-20 (data not shown). Therefore, the candidate peptide antigen should be present in wells 41 and 44, absent in neighboring wells of both microcapillary splits, and either absent or greatly reduced in abundance in subfraction 36-20 where no sensitizing activity was observed. Only a single peptide, that with m/z 854, fulfilled all the above criteria.


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)

Sequential HPLC separation of sensitizing peptides eluted from  the H2-Kb molecule of 8101-RE cells. A shows the results of the firstdimension microbore HPLC separation of peptides eluted from 5 × 1010  8101-RE cells, using HFBA as the ionic modifier. A portion (0.3%) of  each fraction was loaded onto RMA-S cells and tested for lysis by the  anti-A CTL clone in a 51Cr-release assay. Sensitizing fractions 36 and 37  were subfractionated on the same microbore column using TFA as the  ionic modifier (B and C). RMA-S cells were loaded with 1.5% of each  subfraction and tested for lysis by the anti-A CTL clone. The E/T ratio  was 5:1. Peptide loaded RMA-S cell with CTL (solid bars) or without  CTL (hatched bars) as toxicity controls are shown in A. Toxicity controls  were not done in B and C.
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Figure 4: Sequential HPLC separation of sensitizing peptides eluted from the H2-Kb molecule of 8101-RE cells. A shows the results of the firstdimension microbore HPLC separation of peptides eluted from 5 × 1010 8101-RE cells, using HFBA as the ionic modifier. A portion (0.3%) of each fraction was loaded onto RMA-S cells and tested for lysis by the anti-A CTL clone in a 51Cr-release assay. Sensitizing fractions 36 and 37 were subfractionated on the same microbore column using TFA as the ionic modifier (B and C). RMA-S cells were loaded with 1.5% of each subfraction and tested for lysis by the anti-A CTL clone. The E/T ratio was 5:1. Peptide loaded RMA-S cell with CTL (solid bars) or without CTL (hatched bars) as toxicity controls are shown in A. Toxicity controls were not done in B and C.
Mentions: To isolate the naturally processed A antigen peptide, we immunoprecipitated the H-2Kb molecules from 5 × 1010 8101-RE tumor cells. Associated peptides were eluted from the MHC class I molecule with acid, and separated from high molecular mass proteins by filtration through a 5-kD molecular mass cutoff membrane. The unfractionated peptide extract sensitized RMA-S cells for lysis by the anti-A CTL clone (data not shown). The peptide extract was concentrated and then fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) using heptafluorobutyric acid (HFBA) as the ionic modifier. Aliquots from individual fractions were tested. Only two fractions, 36 and 37, sensitized RMA-S cells for lysis by an anti-A CTL clone (Fig. 4 A). Active fractions 36 and 37 were individually rechromatographed over the same HPLC column, using a shallower gradient and TFA as the ionic modifier. A peptide in subfraction 36-19, 36-20 (Fig. 4 B), and 37-19 (Fig. 4 C) sensitized RMA-S cells for lysis by the anti-A CTL clone. However, each subfraction contained more than 100 peptides, too many to sequence with the available material. Therefore, to identify the mass of the A antigen in these mixtures, 60% of each HPLC subfraction was analyzed separately (the remaining 40% was set aside for further analysis) with an on-line microcapillary column effluent splitter, as described previously (33). Four-fifths of the effluent is directed into the mass spectrometer for analysis, while the other one-fifth is simultaneously deposited into a 96-well plate for analysis of sensitizing activity. Since both pieces of data are acquired as a function of time, the ion abundance corresponding to a particular peptide can be correlated with the biological activity. The microcapillary split of subfraction 36-19 and 37-19 yielded activity (Fig. 5, A and B, respectively) but no sensitizing activity was observed from the peptides in subfraction 36-20 (data not shown). Therefore, the candidate peptide antigen should be present in wells 41 and 44, absent in neighboring wells of both microcapillary splits, and either absent or greatly reduced in abundance in subfraction 36-20 where no sensitizing activity was observed. Only a single peptide, that with m/z 854, fulfilled all the above criteria.

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