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

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 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.

Figure 1.

Amino acid sequence of human WT, S20G, and MUT, and RAT amylin. Only the amino acids that differ are shown. The underlined sequence between amino acids 20 to 29 represents the amyloidogenic domain.

Figure 2.

Induction of apoptosis in COS-1 cells expressing intracellular WT and S20G. FACS analysis was performed at 48, 72, and 96 hours using COS-1 cells labeled with AnnexinV-PE after transfection with pMT2 vector (A), pMT2.WT (B), pMT2.S20G (C), pMT2.RAT (D), and pMT2.MUT (E). AnnexinV-PE-positive cells, representing early stage apoptotic cells, are delineated by the bar designated M1.

Figure 3.

Histogram profiles of the induction of apoptosis in COS-1 cells transfected with pMT2 vector (open bars), pMT2.MUT (cross-hatched bars), pMT2.RAT (shaded bars), pMT2.WT (diagonal hatched bars), and pMT2.S20G (solid bars) after 48, 72, and 96 hours. The Annexin-positive and 7-AAD-positive cells representing the late apoptotic or necrotic cells accounted for <5% of the cells in all samples (data not shown). Statistically significant differences are indicated by * (P < 0.05) and ** (P < 0.01). Error bars are standard errors. The data represent the average of seven independent experiments.

Figure 4.

RIA analysis of intracellular WT (solid square), S20G (solid circle), MUT (solid triangle), and RAT (solid diamond) amylin peptide expression in COS-1 cell lysates after transection with the appropriate expression cassette for 48, 72, and 96 hours. Statistically significant differences are denoted by single (P < 0.05) and double (P < 0.01) asterisks. The data represent the average of seven independent experiments. Error bars represent standard errors.

Figure 5.

In vitro generation of amyloid by WT and S20G synthetic peptides as assessed by a ThT binding assay. Open and closed squares represent 10 μmol/L and 25 μmol/L of WT, respectively Open and closed circles represent 10 and 25 μmol/L of S20G, respectively. The data represent the average results from three independent experiments. Error bars represent standard errors.

Figure 6.

Electron microscopy of in vitro-generated amyloid fibers from synthetic WT (A) and S20G (B) amylin. Scale bars, 100 nm.