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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 melanoma tumor lines by D-specific CTLs  (solid symbols) and N-specific CTLs (open symbols) in a 4-h 51Cr-release assay. DM6 (circles), DM93 (squares), and NA8 Mel + Tyr (triangles) express  full-length tyrosinase while NA8 Mel (crosses) does not.
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Figure 2: Recognition of melanoma tumor lines by D-specific CTLs (solid symbols) and N-specific CTLs (open symbols) in a 4-h 51Cr-release assay. DM6 (circles), DM93 (squares), and NA8 Mel + Tyr (triangles) express full-length tyrosinase while NA8 Mel (crosses) does not.

Mentions: To detect either the YMDGTMSQV or the YMNGTMSQV epitopes, we developed CTLs specific for each peptide from transgenic mice expressing a chimeric HLA-A*0201/H2Dd molecule. CTLs generated against the YMDGTMSQV peptide (D-specific CTLs) required 5–6 logs less of the YMDGTMSQV peptide than the YMNGTMSQV peptide to give equivalent lysis of peptide-pulsed target cells (Fig. 1 A). Conversely, CTLs generated against the YMNGTMSQV peptide (N-specific CTLs) required 5–6 logs less YMNGTMSQV than YMDGTMSQV for recognition (Fig. 1 B). Since YMNGTMSQV and YMDGTMSQV bind to HLA-A2.1 with similar affinities (49), these differences reflect the ability of the CTLs to discriminate between these two peptides. Nevertheless, HLA-A*0201+ melanomas expressing tyrosinase were lysed by D-specific CTLs but not by N-specific CTLs at similar effector:target ratios (Fig. 2). The lack of reactivity of N-specific CTLs with these tumors confirms our earlier conclusion based on mass spectrometry data (49) that there is no YMNGTMSQV peptide on the cell surface, although the tyrosinase gene encodes this sequence.


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 melanoma tumor lines by D-specific CTLs  (solid symbols) and N-specific CTLs (open symbols) in a 4-h 51Cr-release assay. DM6 (circles), DM93 (squares), and NA8 Mel + Tyr (triangles) express  full-length tyrosinase while NA8 Mel (crosses) does not.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Recognition of melanoma tumor lines by D-specific CTLs (solid symbols) and N-specific CTLs (open symbols) in a 4-h 51Cr-release assay. DM6 (circles), DM93 (squares), and NA8 Mel + Tyr (triangles) express full-length tyrosinase while NA8 Mel (crosses) does not.
Mentions: To detect either the YMDGTMSQV or the YMNGTMSQV epitopes, we developed CTLs specific for each peptide from transgenic mice expressing a chimeric HLA-A*0201/H2Dd molecule. CTLs generated against the YMDGTMSQV peptide (D-specific CTLs) required 5–6 logs less of the YMDGTMSQV peptide than the YMNGTMSQV peptide to give equivalent lysis of peptide-pulsed target cells (Fig. 1 A). Conversely, CTLs generated against the YMNGTMSQV peptide (N-specific CTLs) required 5–6 logs less YMNGTMSQV than YMDGTMSQV for recognition (Fig. 1 B). Since YMNGTMSQV and YMDGTMSQV bind to HLA-A2.1 with similar affinities (49), these differences reflect the ability of the CTLs to discriminate between these two peptides. Nevertheless, HLA-A*0201+ melanomas expressing tyrosinase were lysed by D-specific CTLs but not by N-specific CTLs at similar effector:target ratios (Fig. 2). The lack of reactivity of N-specific CTLs with these tumors confirms our earlier conclusion based on mass spectrometry data (49) that there is no YMNGTMSQV peptide on the cell surface, although the tyrosinase gene encodes this sequence.

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