Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T cell clones.
The two NOD-derived T cell clones, BDC-2.5 and BDC-6.9, are CD4+, Vbeta4+, islet-specific, and diabetogenic. These two T cell clones show different response patterns to whole islet cell antigen, but were found to respond to the same fraction isolated from beta granule membranes. The clones were used to follow the antigenic activity in the biochemical purification of a beta cell membrane detergent lysate subjected to HPLC anion exchange (IEX) and size exclusion chromatography (SEC). Antigenic activity could be retained after lysis in only one detergent (octyl-beta-glucoside) among several tested. In order to detect solubilized antigen, beta membrane proteins were covalently linked to microlatex beads prior to being added to T cell proliferation assays, a technique that eliminated detergent toxicity and resulted in increased assay sensitivity. To purify the antigen, membrane proteins were absorbed onto an anion exchange column and after elution using a salt gradient, activity for the clones was found in a fraction containing 0.15-0.2 M NaCl. Subsequent analysis of this material by size exclusion chromatography provided an apparent molecular weight of the antigen to be between 50 and 80 kDa. Further attempts to purify the protein by SDS-PAGE resulted in loss of antigenic activity. It is possible that the elusive nature of this protein is a clue to its importance as an autoantigen.
Bergman
B
B
Barbara Davis Center for Childhood Diabetes and Department of Immunology, University of Colorado Health Sciences Center, Denver 80262, USA.
McManaman
J L
JL
Haskins
K
K
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D001324:Autoantigens / Q000276:immunology* / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002999:Clone Cells / Q000276:immunology; D003902:Detergents; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D007515:Islets of Langerhans / Q000737:chemistry / Q000276:immunology*; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*
D000954:Antigens, Surface; D001324:Autoantigens; D003902:Detergents; D018984:Epitopes, T-Lymphocyte; D008565:Membrane Proteins