Abstract
Type 1 diabetes is characterized by the autoimmune destruction of pancreatic β-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 β-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)₂₀₆₋₂₁₄. We identified >2,000 MHC-bound peptides; 1,100 of these presented by β-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP₂₀₆₋₂₁₄ revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1₃₅₅₋₃₆₃ (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 β-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Autoantigens / chemistry
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Autoantigens / isolation & purification
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Autoantigens / metabolism*
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Cell Line
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Chromatography, High Pressure Liquid
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Diabetes Mellitus, Type 1 / immunology
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Diabetes Mellitus, Type 1 / metabolism*
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Female
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Glucose-6-Phosphatase / chemistry
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Glucose-6-Phosphatase / isolation & purification
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Glucose-6-Phosphatase / metabolism
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Histocompatibility Antigens / chemistry
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Histocompatibility Antigens / isolation & purification
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Histocompatibility Antigens / metabolism
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Immunodominant Epitopes / chemistry
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Immunodominant Epitopes / isolation & purification
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Immunodominant Epitopes / metabolism*
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Inflammation Mediators / chemistry
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Inflammation Mediators / isolation & purification
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Inflammation Mediators / metabolism
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Insulin-Secreting Cells / immunology
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Insulin-Secreting Cells / metabolism*
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Interferon-gamma / metabolism
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Janus Kinase 1 / chemistry
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Janus Kinase 1 / isolation & purification
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Janus Kinase 1 / metabolism
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Mice
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Mice, Inbred NOD
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Organ Specificity
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Peptide Fragments / chemistry
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Peptide Fragments / isolation & purification
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Peptide Fragments / metabolism*
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Proteins / chemistry
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Proteins / isolation & purification
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Proteins / metabolism
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Spleen / immunology
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Spleen / metabolism
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Tandem Mass Spectrometry
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Thymus Gland / immunology
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Thymus Gland / metabolism
Substances
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Autoantigens
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Histocompatibility Antigens
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Immunodominant Epitopes
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Inflammation Mediators
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Peptide Fragments
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Proteins
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Interferon-gamma
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Jak1 protein, mouse
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Janus Kinase 1
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Glucose-6-Phosphatase
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G6pc2 protein, mouse