Extended O-GlcNAc on HLA Class-I-Bound Peptides.

We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc's rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane and is hence exposed to glycosyltransferases. We also report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may thus be important targets for immune surveillance.

[1]  Albert J R Heck,et al.  Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry. , 2012, Analytical chemistry.

[2]  David Goldberg,et al.  Lookup peaks: a hybrid of de novo sequencing and database search for protein identification by tandem mass spectrometry. , 2007, Analytical chemistry.

[3]  Albert J R Heck,et al.  Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD) , 2014, Proceedings of the National Academy of Sciences.

[4]  J. Yewdell,et al.  Making sense of mass destruction: quantitating MHC class I antigen presentation , 2003, Nature Reviews Immunology.

[5]  W. Herr,et al.  HCF-1 Is Cleaved in the Active Site of O-GlcNAc Transferase , 2013, Science.

[6]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[7]  M. Mann,et al.  System-Wide Temporal Characterization of the Proteome and Phosphoproteome of Human Embryonic Stem Cell Differentiation , 2011, Science Signaling.

[8]  J. Shabanowitz,et al.  Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis , 2010, Science Signaling.

[9]  H. Rammensee,et al.  Lectin purified human class I MHC-derived peptides: evidence for presentation of glycopeptides in vivo. , 2000, Tissue antigens.

[10]  Lisa M. Ebert,et al.  A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design. , 2009, Cancer research.

[11]  G. Hart,et al.  O-GlcNAc signaling: a metabolic link between diabetes and cancer? , 2010, Trends in biochemical sciences.

[12]  J. Kihlberg,et al.  Immunization with glycosylated Kb‐binding peptides generates carbohydrate‐specific, unrestricted cytotoxic T cells , 1996, European journal of immunology.

[13]  Natalie A Borg,et al.  High Resolution Structures of Highly Bulged Viral Epitopes Bound to Major Histocompatibility Complex Class I , 2005, Journal of Biological Chemistry.

[14]  J. Neefjes,et al.  Into the Intracellular Logistics of Cross-Presentation , 2012, Front. Immun..

[15]  J. McCluskey,et al.  The Impact of a Large and Frequent Deletion in the Human TCR β Locus on Antiviral Immunity , 2012, The Journal of Immunology.

[16]  T. Elliott,et al.  Presentation of Cytosolic Glycosylated Peptides by Human Class I Major Histocompatibility Complex Molecules in Vivo , 1999, The Journal of experimental medicine.

[17]  Natalie A Borg,et al.  T cell receptor recognition of a 'super-bulged' major histocompatibility complex class I–bound peptide , 2005, Nature Immunology.

[18]  T. A. Fritz,et al.  Identification of Common and Unique Peptide Substrate Preferences for the UDP-GalNAc:Polypeptide α-N-acetylgalactosaminyltransferases T1 and T2 Derived from Oriented Random Peptide Substrates* , 2006, Journal of Biological Chemistry.

[19]  Jennifer Couzin-Frankel,et al.  Breakthrough of the year 2013. Cancer immunotherapy. , 2013, Science.

[20]  J. Shabanowitz,et al.  An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins , 1996, The Journal of experimental medicine.

[21]  Carolyn R. Bertozzi,et al.  Essentials of Glycobiology , 1999 .

[22]  Jonas Nilsson,et al.  Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides. , 2014, Journal of proteome research.

[23]  J. Neefjes,et al.  Recycling MHC class I molecules and endosomal peptide loading. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Hongfang Liu,et al.  dbOGAP - An Integrated Bioinformatics Resource for Protein O-GlcNAcylation , 2011, BMC Bioinformatics.

[25]  Francesco M Marincola,et al.  Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going? , 2002, Journal of the National Cancer Institute.

[26]  G. Hart,et al.  Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. , 2011, Annual review of biochemistry.

[27]  N. Callewaert,et al.  GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins , 2014, Nature Biotechnology.

[28]  L. Otvos,et al.  Complex Carbohydrates Are Not Removed During Processing of Glycoproteins by Dendritic Cells , 2002, The Journal of experimental medicine.

[29]  Karl Mechtler,et al.  Unambiguous Phosphosite Localization using Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) , 2013, Journal of proteome research.

[30]  Jennifer G. Abelin,et al.  MHC Class I–Associated Phosphopeptides Are the Targets of Memory-like Immunity in Leukemia , 2013, Science Translational Medicine.

[31]  M. Grégoire,et al.  MUC1-Specific Cytotoxic T Lymphocytes in Cancer Therapy: Induction and Challenge , 2012, BioMed research international.

[32]  H. Rammensee,et al.  Autophagy promotes MHC class II presentation of peptides from intracellular source proteins , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Wolfert,et al.  Adaptive immune activation: glycosylation does matter. , 2013, Nature chemical biology.