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Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo.

Haurum JS, Høier IB, Arsequell G, Neisig A, Valencia G, Zeuthen J, Neefjes J, Elliott T - J. Exp. Med. (1999)

Bottom Line: In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide.Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum.Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Radcliffe Hospital, Oxford, England. haurum@cancer.dk

ABSTRACT
Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8-10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-beta-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-beta-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

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TAP-mediated transport of posttranslationally modified peptides. (A) Competition of peptide 417 translocation by glycopeptide wt-G (○), unmodified peptide wt-S (▵), or index peptide 417 (▴). The amount of iodinated index peptide is expressed as a fraction of the amount recovered in the absence of competitor. (B) Direct translocation of iodinated glycopeptides was analyzed by comparing translocation of peptide 417 with a serine-substituted analogue (417-S), as well as an O-GlcNAc–glycosylated (417-G) version thereof. The results are the amounts of translocated peptide recovered expressed as a fraction of total input peptide.
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Figure 1: TAP-mediated transport of posttranslationally modified peptides. (A) Competition of peptide 417 translocation by glycopeptide wt-G (○), unmodified peptide wt-S (▵), or index peptide 417 (▴). The amount of iodinated index peptide is expressed as a fraction of the amount recovered in the absence of competitor. (B) Direct translocation of iodinated glycopeptides was analyzed by comparing translocation of peptide 417 with a serine-substituted analogue (417-S), as well as an O-GlcNAc–glycosylated (417-G) version thereof. The results are the amounts of translocated peptide recovered expressed as a fraction of total input peptide.

Mentions: Assays for the TAP-mediated translocation of radiolabeled, posttranslationally modified peptides across the ER membrane of human LCL721 cells were performed as described 13. T2 cells were used to demonstrate the TAP dependence of transport. Samples incubated in the absence of ATP were carried out as controls for the ATP dependence of the transport. In competition assays, iodinated peptide 417 was mixed with competitor peptide before addition to permeabilized cells. The substrate peptide 417-G carrying O-β-GlcNAc monosaccharide is not itself a ligand for Con A in the absence of an N-linked glycan, as seen from the inability to recover 417-G by Con A–Sepharose in the absence of ATP (see Fig. 1 B).


Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo.

Haurum JS, Høier IB, Arsequell G, Neisig A, Valencia G, Zeuthen J, Neefjes J, Elliott T - J. Exp. Med. (1999)

TAP-mediated transport of posttranslationally modified peptides. (A) Competition of peptide 417 translocation by glycopeptide wt-G (○), unmodified peptide wt-S (▵), or index peptide 417 (▴). The amount of iodinated index peptide is expressed as a fraction of the amount recovered in the absence of competitor. (B) Direct translocation of iodinated glycopeptides was analyzed by comparing translocation of peptide 417 with a serine-substituted analogue (417-S), as well as an O-GlcNAc–glycosylated (417-G) version thereof. The results are the amounts of translocated peptide recovered expressed as a fraction of total input peptide.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195561&req=5

Figure 1: TAP-mediated transport of posttranslationally modified peptides. (A) Competition of peptide 417 translocation by glycopeptide wt-G (○), unmodified peptide wt-S (▵), or index peptide 417 (▴). The amount of iodinated index peptide is expressed as a fraction of the amount recovered in the absence of competitor. (B) Direct translocation of iodinated glycopeptides was analyzed by comparing translocation of peptide 417 with a serine-substituted analogue (417-S), as well as an O-GlcNAc–glycosylated (417-G) version thereof. The results are the amounts of translocated peptide recovered expressed as a fraction of total input peptide.
Mentions: Assays for the TAP-mediated translocation of radiolabeled, posttranslationally modified peptides across the ER membrane of human LCL721 cells were performed as described 13. T2 cells were used to demonstrate the TAP dependence of transport. Samples incubated in the absence of ATP were carried out as controls for the ATP dependence of the transport. In competition assays, iodinated peptide 417 was mixed with competitor peptide before addition to permeabilized cells. The substrate peptide 417-G carrying O-β-GlcNAc monosaccharide is not itself a ligand for Con A in the absence of an N-linked glycan, as seen from the inability to recover 417-G by Con A–Sepharose in the absence of ATP (see Fig. 1 B).

Bottom Line: In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide.Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum.Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Radcliffe Hospital, Oxford, England. haurum@cancer.dk

ABSTRACT
Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8-10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-beta-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-beta-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

Show MeSH
Related in: MedlinePlus