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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 the melanoma line DM93 by D Tyr CTL.  DM93 was left untreated (crosses) or acid-treated as described in Materials  and Methods and then incubated at 37°C for 5 h either in the absence of  any inhibitor (diamonds) or in the presence of one of the follwing: 10 μg/ml  BFA (squares), 20 μM lactacystin (triangles) or 20 μM LLnL (circles). All  targets were then washed and used in a 4-h 51Cr-release assay in the presence of 10 μg/ml BFA to block any further expression of newly synthesized peptide–HLA-A*0201 complexes. These results are representative of  four independent experiments.
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Figure 8: Recognition of the melanoma line DM93 by D Tyr CTL. DM93 was left untreated (crosses) or acid-treated as described in Materials and Methods and then incubated at 37°C for 5 h either in the absence of any inhibitor (diamonds) or in the presence of one of the follwing: 10 μg/ml BFA (squares), 20 μM lactacystin (triangles) or 20 μM LLnL (circles). All targets were then washed and used in a 4-h 51Cr-release assay in the presence of 10 μg/ml BFA to block any further expression of newly synthesized peptide–HLA-A*0201 complexes. These results are representative of four independent experiments.

Mentions: Because of the TAP dependence of the YMDGTMSQV epitope, we were interested in determining whether the proteasome is involved in epitope formation. Proteasome-dependent processing was evaluated by brief exposure of target cells to pH 2.5 medium to denature all cell surface MHC class I molecules, followed by incubation to allow reexpression of the epitope in the presence or absence of lactacystin or LLnL as described elsewhere (Luckey, C.J., G.M. King, J.A. Marto, B.F. Maier, V.L. Crotzer, T.A. Colella, J. Shabanowitz, D.F. Hunt, and V.H. Engelhard, manuscript submitted for publication). CTL recognition of acid-treated target cells was abolished, but recovered to control levels if the targets were incubated for 5 h at 37°C (Fig. 8). This recovery of epitope expression was inhibited by >50% in the presence of BFA, which blocks the egress of newly synthesized peptide MHC class I complexes from the ER (66–68). Although the failure of BFA to completely inhibit reexpression of the epitope is not understood, the difference in recognition between the acid-washed, untreated cells and the acid-washed, BFA-treated cells allowed us to determine whether protease inhibitors also blocked the generation of the YMDGTMSQV epitope. Incubation of acid-washed targets with 20 μM LLnL, a concentration known to selectively inhibit the proteasome (59), inhibited reexpression of the YMDGTMSQV epitope on the cell surface to the same extent as BFA (Fig. 8). In addition, treatment of the acid-washed cells with lactacystin, a highly specific inhibitor of the proteasome (58), also blocked reexpression of the YMDGTMSQV epitope. It has been shown elsewhere that the effects of these proteasome inhibitors are not due to nonspecific effects on class I biosynthesis or susceptibility of target cells to CTL-mediated lysis (Luckey, C.M.J., G.M. King, J.A. Marto, B.F. Maier, V.L. Crotzer, T.A. Colella, J. Shabanowitz, D.F. Hunt, and V.H. Engelhard, manuscript submitted for publication). Together these results indicate that the processing of intact tyrosinase to generate the YMDGTMSQV epitope is dependent upon the proteasome.


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 the melanoma line DM93 by D Tyr CTL.  DM93 was left untreated (crosses) or acid-treated as described in Materials  and Methods and then incubated at 37°C for 5 h either in the absence of  any inhibitor (diamonds) or in the presence of one of the follwing: 10 μg/ml  BFA (squares), 20 μM lactacystin (triangles) or 20 μM LLnL (circles). All  targets were then washed and used in a 4-h 51Cr-release assay in the presence of 10 μg/ml BFA to block any further expression of newly synthesized peptide–HLA-A*0201 complexes. These results are representative of  four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 8: Recognition of the melanoma line DM93 by D Tyr CTL. DM93 was left untreated (crosses) or acid-treated as described in Materials and Methods and then incubated at 37°C for 5 h either in the absence of any inhibitor (diamonds) or in the presence of one of the follwing: 10 μg/ml BFA (squares), 20 μM lactacystin (triangles) or 20 μM LLnL (circles). All targets were then washed and used in a 4-h 51Cr-release assay in the presence of 10 μg/ml BFA to block any further expression of newly synthesized peptide–HLA-A*0201 complexes. These results are representative of four independent experiments.
Mentions: Because of the TAP dependence of the YMDGTMSQV epitope, we were interested in determining whether the proteasome is involved in epitope formation. Proteasome-dependent processing was evaluated by brief exposure of target cells to pH 2.5 medium to denature all cell surface MHC class I molecules, followed by incubation to allow reexpression of the epitope in the presence or absence of lactacystin or LLnL as described elsewhere (Luckey, C.J., G.M. King, J.A. Marto, B.F. Maier, V.L. Crotzer, T.A. Colella, J. Shabanowitz, D.F. Hunt, and V.H. Engelhard, manuscript submitted for publication). CTL recognition of acid-treated target cells was abolished, but recovered to control levels if the targets were incubated for 5 h at 37°C (Fig. 8). This recovery of epitope expression was inhibited by >50% in the presence of BFA, which blocks the egress of newly synthesized peptide MHC class I complexes from the ER (66–68). Although the failure of BFA to completely inhibit reexpression of the epitope is not understood, the difference in recognition between the acid-washed, untreated cells and the acid-washed, BFA-treated cells allowed us to determine whether protease inhibitors also blocked the generation of the YMDGTMSQV epitope. Incubation of acid-washed targets with 20 μM LLnL, a concentration known to selectively inhibit the proteasome (59), inhibited reexpression of the YMDGTMSQV epitope on the cell surface to the same extent as BFA (Fig. 8). In addition, treatment of the acid-washed cells with lactacystin, a highly specific inhibitor of the proteasome (58), also blocked reexpression of the YMDGTMSQV epitope. It has been shown elsewhere that the effects of these proteasome inhibitors are not due to nonspecific effects on class I biosynthesis or susceptibility of target cells to CTL-mediated lysis (Luckey, C.M.J., G.M. King, J.A. Marto, B.F. Maier, V.L. Crotzer, T.A. Colella, J. Shabanowitz, D.F. Hunt, and V.H. Engelhard, manuscript submitted for publication). Together these results indicate that the processing of intact tyrosinase to generate the YMDGTMSQV epitope is dependent upon the proteasome.

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