S20G mutant amylin exhibits increased in vitro amyloidogenicity and increased intracellular cytotoxicity compared to wild-type amylin

Citation
S. Sakagashira et al., S20G mutant amylin exhibits increased in vitro amyloidogenicity and increased intracellular cytotoxicity compared to wild-type amylin, AM J PATH, 157(6), 2000, pp. 2101-2109
Citations number
36
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology","Medical Research Diagnosis & Treatment
Journal title
AMERICAN JOURNAL OF PATHOLOGY
ISSN journal
00029440 → ACNP
Volume
157
Issue
6
Year of publication
2000
Pages
2101 - 2109
Database
ISI
SICI code
0002-9440(200012)157:6<2101:SMAEII>2.0.ZU;2-Q
Abstract
Human amylin, a major constituent of pancreatic amyloid deposits, may be a pathogenetic factor for noninsulin-dependent diabetes mellitus (NIDDM). We demonstrated that the human amylin S20G gene mutation (S20G) was associated with a history of early onset, more severe type of NIDDM, linking the amyl in gene to this disease. Also, we demonstrated that expression of human wil d-type (WT) amylin in COS-1 cells leads to intracellular amyloidogenesis an d induction of apoptosis, suggesting a possible mechanism for disease induc tion, Therefore we compared the abilities of S20G and WT amylin to induce a poptosis in transfected COS-1 cells and form amyloid in vitro. We transfect ed the rat(RAT), mutated human (MUT), WT, and S20G amylin genes into COS-1 cells and measured apoptosis using fluorescent-activate cell sorting analys is at 48, 72, and 96 hours. At 96 hours apoptosis increased significantly ( P < 0.01) in cells transfected with WT and S20G over RAT or MUT (WT, 19%; S 20G, 25%; RAT, 13%; and MUT, 12%) and the difference between WT and S20G wa s significant (P < 0.05). Synthetic WT and S20G monomeric peptides were use d to generate amyloid fibrils in vitro as measured by the thioflavin T bind ing assay, The S20G amylin formed approximately twofold more amyloid at a r ate approximately threefold higher than WT. Electron micrography indicated that the in vitro amyloid generated by WT and S20G amylins were morphologic ally indistinguishable. The results suggest that increased cytotoxicity by S20G is because of increased amyloidogenicity, which may be a causative fac tor in the early development of NIDDM, possibly through loss of beta cell m ass.