Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.
IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.
Xie
H
H
Oral Infection and Immunity Branch, National Institute of Dental Research, Bethesda, MD 20892, USA.
Zhang
B
B
Matsumoto
Y
Y
Li
Q
Q
Notkins
A L
AL
Lan
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000737:chemistry* / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology*; D001666:Binding Sites, Antibody; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D018604:Epitope Mapping; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D001323:Autoantibodies; D001324:Autoantigens; D004220:Disulfides; D008565:Membrane Proteins; D010446:Peptide Fragments; C516862:PTPRN protein, human; C102889:PTPRN2 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516863:Ptprn protein, mouse; C516864:Ptprn protein, rat; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D014357:Trypsin