In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH.
Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.
Hampl
J
J
Institut für Immunologie der Joh. Gutenberg Universität, Mainz, FRG.
Gradehandt
G
G
Kalbacher
H
H
Rüde
E
E
D000818:Animals; D000938:Antigen-Presenting Cells / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000737:chemistry* / Q000276:immunology; D007377:Interleukin-3 / Q000032:analysis; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D013601:T-Lymphocytes / Q000276:immunology*
D007328:Insulin; D007377:Interleukin-3; D008623:Mercaptoethanol; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol