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The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.

Mosse CA, Meadows L, Luckey CJ, Kittlesen DJ, Huczko EL, Slingluff CL, Shabanowitz J, Hunt DF, Engelhard VH - J. Exp. Med. (1998)

Bottom Line: Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum.Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent.We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22904, USA.

ABSTRACT
Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

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Recognition of NA8 Mel and transfectants by N-specific (A)  and D-specific (B) CTLs in a 4-h 51Cr-release assay. Targets used were  the tyrosinase negative melanoma NA8 Mel (circles), its full-length tyrosinase transfectant NA8 Mel + Tyr (squares), or its T3.1 transfectant NA8  Mel + T3.1 (triangles). The results are representative of five independent  experiments.
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Figure 5: Recognition of NA8 Mel and transfectants by N-specific (A) and D-specific (B) CTLs in a 4-h 51Cr-release assay. Targets used were the tyrosinase negative melanoma NA8 Mel (circles), its full-length tyrosinase transfectant NA8 Mel + Tyr (squares), or its T3.1 transfectant NA8 Mel + T3.1 (triangles). The results are representative of five independent experiments.

Mentions: One explanation for the exclusive production of YMDGTMSQV from full-length tyrosinase and of YMN from the minigene is that the former is translated in the ER, where it undergoes conversion, whereas the latter is translated in the cytosol, where it does not. However, the minigene product was preprocessed to the ideal length to bind to HLA-A*0201, while the full-length tyrosinase requires proteolysis before binding. Therefore, it was also possible that immediate binding of the minigene product to HLA-A*0201 after entry into the ER prevented conversion, whereas longer peptides derived from full-length tyrosinase were converted before being trimmed to the optimal length for binding. To distinguish between these two possibilities, we transfected NA8 Mel, a tyrosinase-negative melanoma cell line, with a truncated tyrosinase gene fragment called T3.1 (51). The translation product of T3.1 spans residues 143–377 of the full-length tyrosinase sequence (Fig. 4). Because it lacks the NH2-terminal end of full-length tyrosinase, including the signal sequence, it should be translated in the cytosol, as is the minigene, rather than the ER. However, the YMNGTMSQV epitope sequence in T3.1 (residues 368–376) is bounded by 225 residues at its NH2 terminus and 1 residue at its COOH terminus. To be presented by HLA-A*0201, it requires additional processing as compared to the minigene product. If the failure of the minigene product to undergo conversion were due to its expression in the cytosol, regardless of length, then we would expect that cells expressing T3.1 would also express the unconverted peptide epitope. This was found to be the case (Fig. 5 A). We conclude from this that the production of the unconverted epitope is associated with translation in the cytosol, and that its absence from cells expressing full-length tyrosinase indicates that the converted epitope is produced from this protein after it is translated into the ER, rather than mistranslated in the cytosol. Although the presence of the unconverted epitope was clearly associated with the translation of protein in the cytosol, the absence of the converted epitope was not. Surprisingly, we found that cells expressing T3.1 presented the converted epitope as well as the unconverted form (Fig. 5 B). This low but reproducible level of lysis by the D-specific CTL indicates that cytosolic proteins can give rise to epitopes that have undergone conversion.


The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.

Mosse CA, Meadows L, Luckey CJ, Kittlesen DJ, Huczko EL, Slingluff CL, Shabanowitz J, Hunt DF, Engelhard VH - J. Exp. Med. (1998)

Recognition of NA8 Mel and transfectants by N-specific (A)  and D-specific (B) CTLs in a 4-h 51Cr-release assay. Targets used were  the tyrosinase negative melanoma NA8 Mel (circles), its full-length tyrosinase transfectant NA8 Mel + Tyr (squares), or its T3.1 transfectant NA8  Mel + T3.1 (triangles). The results are representative of five independent  experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199193&req=5

Figure 5: Recognition of NA8 Mel and transfectants by N-specific (A) and D-specific (B) CTLs in a 4-h 51Cr-release assay. Targets used were the tyrosinase negative melanoma NA8 Mel (circles), its full-length tyrosinase transfectant NA8 Mel + Tyr (squares), or its T3.1 transfectant NA8 Mel + T3.1 (triangles). The results are representative of five independent experiments.
Mentions: One explanation for the exclusive production of YMDGTMSQV from full-length tyrosinase and of YMN from the minigene is that the former is translated in the ER, where it undergoes conversion, whereas the latter is translated in the cytosol, where it does not. However, the minigene product was preprocessed to the ideal length to bind to HLA-A*0201, while the full-length tyrosinase requires proteolysis before binding. Therefore, it was also possible that immediate binding of the minigene product to HLA-A*0201 after entry into the ER prevented conversion, whereas longer peptides derived from full-length tyrosinase were converted before being trimmed to the optimal length for binding. To distinguish between these two possibilities, we transfected NA8 Mel, a tyrosinase-negative melanoma cell line, with a truncated tyrosinase gene fragment called T3.1 (51). The translation product of T3.1 spans residues 143–377 of the full-length tyrosinase sequence (Fig. 4). Because it lacks the NH2-terminal end of full-length tyrosinase, including the signal sequence, it should be translated in the cytosol, as is the minigene, rather than the ER. However, the YMNGTMSQV epitope sequence in T3.1 (residues 368–376) is bounded by 225 residues at its NH2 terminus and 1 residue at its COOH terminus. To be presented by HLA-A*0201, it requires additional processing as compared to the minigene product. If the failure of the minigene product to undergo conversion were due to its expression in the cytosol, regardless of length, then we would expect that cells expressing T3.1 would also express the unconverted peptide epitope. This was found to be the case (Fig. 5 A). We conclude from this that the production of the unconverted epitope is associated with translation in the cytosol, and that its absence from cells expressing full-length tyrosinase indicates that the converted epitope is produced from this protein after it is translated into the ER, rather than mistranslated in the cytosol. Although the presence of the unconverted epitope was clearly associated with the translation of protein in the cytosol, the absence of the converted epitope was not. Surprisingly, we found that cells expressing T3.1 presented the converted epitope as well as the unconverted form (Fig. 5 B). This low but reproducible level of lysis by the D-specific CTL indicates that cytosolic proteins can give rise to epitopes that have undergone conversion.

Bottom Line: Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum.Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent.We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22904, USA.

ABSTRACT
Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

Show MeSH
Related in: MedlinePlus