Human Monoclonal Antibodies Isolated from Type I Diabetes Patients Define Multiple Epitopes in the Protein Tyrosine Phosphatase-Like IA-2 Antigen

Abstract

Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2β-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777–979. Two of these hAbs cross-reacted with the related IA-2β PTP-like domain (IA-2β aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687–979) for binding, but critical amino acids clustered in the N-terminal region 687–777. An additional epitope could be localized within the juxtamembrane domain (aa 603–779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a VH germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven.

Autoantibodies in Diabetes

“…IA-2Ab epitopes have been mapped using different approaches including the closely related IA-2␤ (70 -73), fusion proteins of IA-2 and IA-2␤ (74,75), and human monoclonal antibodies specific to IA-2 (76,77). IA-2 appears to be the primary autoantigen in type 1 diabetes, whereas antibodies to IA-2␤ are considered to be the result of subsequent epitope spreading (8,70,75,78).…”

“…High susceptibility HLA genotypes were associated with the presence of multiple epitopes to both IA-2 and IA-2␤, but not with IA-2Ab specific to the juxtamembrane region (78,80). In an effort to better characterize the conformational epitopes, competition assays with human monoclonal antibodies were developed (76). A major conformational epitope located at the COOH-terminal region of the protein tyrosine phosphatase domain was identified using this approach (76,77).…”

“…In an effort to better characterize the conformational epitopes, competition assays with human monoclonal antibodies were developed (76). A major conformational epitope located at the COOH-terminal region of the protein tyrosine phosphatase domain was identified using this approach (76,77).…”

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…Purified rFab KSAb1 or anti-TSHR mAbs were used as a positive control and rFab 96/3, specific for a pancreatic islet cell Ag IA-2, served as a negative control (33) for the bacterial produced Abs. Radioligand binding inhibition assay.…”

Identification of Tyrosine Phosphatase 2(256–760) Construct as a New, Sensitive Marker for the Detection of Islet Autoimmunity in Type 2 Diabetic Patients

“…The finding that IA-2 (256 -760) construct reacts with more LADA patients than IA-2 FL (1-979) , even if the former is a portion of the latter, might be due to a different conformation of the 256 -760 amino acidic residues of the two constructs or to steric hindrances, ultimately leading to variable autoantibody binding affinities. Several studies demonstrated that IA-2As in type 1 diabetic patients are directed against multiple epitopes of the intracellular cytoplasmic portion of the protein (amino acids 601-979), located in the juxtamembrane region (JM, amino acids 605-682), and in the protein (PTP)-like COOH-terminal domain (amino acids 630 -979) (31)(32)(33)(34)(35)(36). To date, an immune response against the extracellular domain of the IA-2 protein has not been demonstrated in type 1 diabetes.…”