S20G mutant amylin exhibits increased in vitro amyloidogenicity and increased intracellular cytotoxicity compared to wild-type amylin.
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 amylin gene to this disease. Also, we demonstrated that expression of human wild-type (WT) amylin in COS-1 cells leads to intracellular amyloidogenesis and induction of apoptosis, suggesting a possible mechanism for disease induction. Therefore we compared the abilities of S20G and WT amylin to induce apoptosis in transfected COS-1 cells and form amyloid in vitro. We transfected the rat (RAT), mutated human (MUT), WT, and S20G amylin genes into COS-1 cells and measured apoptosis using fluorescent-activated cell sorting analysis 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%; S20G, 25%; RAT, 13%; and MUT, 12%) and the difference between WT and S20G was significant (P < 0.05). Synthetic WT and S20G monomeric peptides were used to generate amyloid fibrils in vitro as measured by the thioflavin T binding assay. The S20G amylin formed approximately twofold more amyloid at a rate approximately threefold higher than WT. Electron micrography indicated that the in vitro amyloid generated by WT and S20G amylins were morphologically indistinguishable. The results suggest that increased cytotoxicity by S20G is because of increased amyloidogenicity, which may be a causative factor in the early development of NIDDM, possibly through loss of ss cell mass.
Sakagashira
S
S
Departments of Medicine, Biochemistry and Molecular Biology, and Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.
Hiddinga
H J
HJ
Tateishi
K
K
Sanke
T
T
Hanabusa
T
T
Nanjo
K
K
Eberhardt
N L
NL
D000682:Amyloid / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology* / Q000648:ultrastructure; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019556:COS Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects*; D058228:Islet Amyloid Polypeptide; D008854:Microscopy, Electron; D009154:Mutation; D051381:Rats; D012016:Reference Values; D013997:Time Factors; D014162:Transfection
D000682:Amyloid; D058228:Islet Amyloid Polypeptide