IDDM Patients' Sera Recognize a Novel 30-KD Pancreatic Autoantigen Related to Chymotrypsinogen

Abstract

We have examined, by western immunoblot analysis, the sera of 16 insulin-dependent diabetes mellitus patients (IDDM) for the presence of autoantibodies against proteins extracted from islet-cell enriched preparations of normal human pancreata. A novel putative autoantigen recognized by late stage IDDM patients sera was identified, and its amino acid sequence was partially determined. Islets of Langerhans were partially purified by a modified collagenase digestion procedure, and subsequent protein extracts were fractionated by one-dimensional or two-dimensional polyacrylamide gel electrophoresis (1-D or 2-D SDS-PAGE). Immunoblot analysis revealed a 30-kD species which was recognized by 4 of 16 IDDM patients sera, but none of 16 normal sera. The 30-kD protein, appeared as a single band on 1-D SDS-PAGE, but was resolved on 2-D gel electrophoresis as several distinct protein species with different isoelectric points (pI's), ranging from 7 to 9. The amino terminal sequence of one such species was partially determined by microsequencing, and the second through the fourteenth amino acids were found to be identical to the corresponding sequence in human chymotrypsinogen. The fifteenth through the eighteenth amino acids were different from the known chymotrypsinogen sequence. This region corresponds with the site that is cleaved to activate chymotrypsinogen. Based on the size and sequence homology, this antigen appears to be related to chymotrypsinogen. We conclude that this 30-kD species may be an autoantigen in some late stage IDDM patients.

The pancreatic β cell and type 1 diabetes: innocent bystander or active participant?

“…In T1DM patients, mild pancreatic exocrine deficiency not requiring pancreatic enzyme replacement is noted in up to 80% of patients [25]; however, a decade later the mild exocrine insufficiency resolved, suggesting that exocrine deficiency represents an early defect following T1DM onset [26]. Chymotrypsinogen is also an autoantigen in patients with T1DM [27]. It is intriguing to speculate that these observations relate to the anatomic and functional relationship between islets and the surrounding acinar parenchyma [28].…”

Novel Biomarkers in Type 1 Diabetes

“…Today, the most useful proteomic biomarkers for T1D are "islet autoantibodies" present in serum, which are strong predictors of the later development of T1D. Since the 1970s, a series of islet autoantibodies (over 20) involved in T1D has been discovered [60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79]. Autoantibody assays have constantly been improved.…”

Islet protein profiling

“…Identification of a protein can be obtained by revealing its amino acid sequence. As this is a time-consuming procedure, the approach has only been used in one study in islet protein profiling [21]. Instead of sequencing tryptic peptides obtained from proteins isolated from islets or islet cells, accurate mass determination by tandem mass spectrometry [74] of the tryptic peptides allowed their comparison with known tryptic cleavage products of proteins contained in protein databases.…”