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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 RNA helicase peptide was generated by a single amino acid substitution that resulted from a single nucleotide change  in 8101-RE. cDNA sequences of murine p68 RNA helicase are compared with p68 sequences of 8101-RE in the region of the mutation. Sequence identity is indicated by ---. RE: sequences from 4/6 cDNA  clones from 8101-RE. HLF: sequence from 6 cDNA clones from 8101HLF. WT: published murine p68 RNA helicase cDNA sequence (reference 38). A single nucleotide substitution of C→ T at nucleotide 1812  was found in the 8101-RE tumor, but not in 8101-HLF.
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Figure 7: The mutant p68 RNA helicase peptide was generated by a single amino acid substitution that resulted from a single nucleotide change in 8101-RE. cDNA sequences of murine p68 RNA helicase are compared with p68 sequences of 8101-RE in the region of the mutation. Sequence identity is indicated by ---. RE: sequences from 4/6 cDNA clones from 8101-RE. HLF: sequence from 6 cDNA clones from 8101HLF. WT: published murine p68 RNA helicase cDNA sequence (reference 38). A single nucleotide substitution of C→ T at nucleotide 1812 was found in the 8101-RE tumor, but not in 8101-HLF.

Mentions: The peptide SNFVFAGI was analyzed for homology with known protein sequences using the BLAST program (37). The tumor-derived peptide matched the murine p68 RNA helicase sequence (38) except that the former had phenylalanine instead of serine at position five, suggesting that the tumor peptide might be encoded by a mutant p68 RNA helicase gene in the tumor cells. To confirm this hypothesis cDNA was synthesized and amplified from tumor cell (8101-RE) mRNA by RT-PCR and primers specific for the p68 RNA helicase. The amplified 2.1-kb product pooled from three independent RT-PCR reactions was cloned into the vector pcDNA3. Six cDNA clones were sequenced using primers for the 3′ end of the insert, that included the region of the putative mutation. Two of the six clones were identical to the wild-type sequence of murine p68 RNA (38) helicase while the other four had a T instead of a C at the nucleotide position 1812. This nucleotide substitution resulted in a change to phenylalanine from serine at amino acid 551 (Fig. 7). The C to T transition, which occurred at a dipyrimidine site, is a commonly observed UV-induced mutation (39).


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 RNA helicase peptide was generated by a single amino acid substitution that resulted from a single nucleotide change  in 8101-RE. cDNA sequences of murine p68 RNA helicase are compared with p68 sequences of 8101-RE in the region of the mutation. Sequence identity is indicated by ---. RE: sequences from 4/6 cDNA  clones from 8101-RE. HLF: sequence from 6 cDNA clones from 8101HLF. WT: published murine p68 RNA helicase cDNA sequence (reference 38). A single nucleotide substitution of C→ T at nucleotide 1812  was found in the 8101-RE tumor, but not in 8101-HLF.
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Related In: Results  -  Collection

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Figure 7: The mutant p68 RNA helicase peptide was generated by a single amino acid substitution that resulted from a single nucleotide change in 8101-RE. cDNA sequences of murine p68 RNA helicase are compared with p68 sequences of 8101-RE in the region of the mutation. Sequence identity is indicated by ---. RE: sequences from 4/6 cDNA clones from 8101-RE. HLF: sequence from 6 cDNA clones from 8101HLF. WT: published murine p68 RNA helicase cDNA sequence (reference 38). A single nucleotide substitution of C→ T at nucleotide 1812 was found in the 8101-RE tumor, but not in 8101-HLF.
Mentions: The peptide SNFVFAGI was analyzed for homology with known protein sequences using the BLAST program (37). The tumor-derived peptide matched the murine p68 RNA helicase sequence (38) except that the former had phenylalanine instead of serine at position five, suggesting that the tumor peptide might be encoded by a mutant p68 RNA helicase gene in the tumor cells. To confirm this hypothesis cDNA was synthesized and amplified from tumor cell (8101-RE) mRNA by RT-PCR and primers specific for the p68 RNA helicase. The amplified 2.1-kb product pooled from three independent RT-PCR reactions was cloned into the vector pcDNA3. Six cDNA clones were sequenced using primers for the 3′ end of the insert, that included the region of the putative mutation. Two of the six clones were identical to the wild-type sequence of murine p68 RNA (38) helicase while the other four had a T instead of a C at the nucleotide position 1812. This nucleotide substitution resulted in a change to phenylalanine from serine at amino acid 551 (Fig. 7). The C to T transition, which occurred at a dipyrimidine site, is a commonly observed UV-induced mutation (39).

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