Peptide mapping and characterisation of glycation patterns of the glima 38 antigen recognised by autoantibodies in Type I diabetic patients.
OBJECTIVE
Glima 38 is an N-glycated neuroendocrine membrane protein of M(r) 38,000, which is recognised by autoantibodies in approximately 20% of patients with Type I (insulin-dependent) diabetes mellitus. The aim of this study was to characterise the carbohydrate moiety and generate peptide maps of glima 38.


METHODS
Sera of high immunoreactivity to glima 38 were used to isolate 35-S methionine-labelled protein from betaTC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidases. The protein moiety was subjected to digestion by proteases to define peptide maps.


RESULTS
The autoreactive epitopes in glima 38 recognised by Type I diabetic sera are conformational and independent of the carbohydrate moiety. Inhibition of N-glycation of glima 38 in vivo, shows a protein core of M(r) 22,000 in both pancreatic beta-(betaTC3) and neuronal (GT1.7) cell lines. The carbohydrate moieties in the two cell types are distinct but contain a similar amount of terminal sialic acid residues and at least five oligosaccharide chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. Endoproteinase treatment of the M(r) 22,000 core protein results in peptides of M(r) 4500 and M(r) 20,000 with Lys-C, and peptides of M(r) 4000 and M(r) 11,000-12,000 with Glu-C/V8 and Asp-N proteases.


CONCLUSIONS
The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purification.
Roll
U
U
Department of Medicine, Hormone Research Institute, University of California San Francisco, USA.
Turck
C W
CW
Gitelman
S E
SE
Rosenthal
S M
SM
Nolte
M S
MS
Masharani
U
U
Ziegler
A G
AG
Baekkeskov
S
S
D000293:Adolescent; D000328:Adult; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D002846:Chromatography, Affinity; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D007223:Infant; D007515:Islets of Langerhans / Q000276:immunology*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
D001323:Autoantibodies; D001324:Autoantigens; D008565:Membrane Proteins; D010446:Peptide Fragments; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D010450:Endopeptidases