11012
J. Am. Chem. SOC.1993, 115, 11012-11013
Interspecies Sequence Variations Affect the Kinetics and Thermodynamics of Amyloid Formation: Peptide Models of Pancreatic Amyloid
1
20
37
IAPP"
ACHN-SNNFGAILSS-CONHZ
Ted T. Ashburn and Peter T. Lansbury, Jr.' Department of Chemistry Massachusetts Institute of Technology Cambridge, Massachusetts 02139
29
HzN-KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNW-CONH?
IAPPH(20-29) c\
Yp
ACHN-SNNLGAILSS-CONHZ
ACHN-SNNFGAILSP-CONH,
IAPPH(20-29)F23L
IAPPH(20-29)S29P
Received June 2. 1993
Human pancreatic amyloid deposits comprising the islet amyloid polypeptide (IAPPH, Figure 1) are characteristic of type I1 diabetes.1 A 10 amino acid sequence within IAPPH (residues 20-29) is sufficient for amyloid f o r m a t i ~ n . ~We . ~ proposed a model for the IAPPH(20-29) peptide amyloid based on electron microscopy (EM),3 Fourier-transform infrared spectroscopy (FTIR), and solid-state nuclear magnetic resonance spectroscopy (ssNMR) studies,4 in which the GAIL5 sequence (residues 2427) forms an ordered antiparallel @-sheet,while the termini of the peptide have a less regular and possibly more flexible ~ t r u c t u r e .A~ peptide based on the rat IAPP(20-29) sequence, which differs from IAPPH at positions 23, 25, 26, 28, and 29, is not amyloidogenic.6 However, a peptide based on the cat IAPP sequence (IAPF(20-29), Figure l), which differs from the human sequence at positions 23 (Leu vs Phe) and 29 (Pro vs Ser), forms amyloid fibrils in vitro.3.' We report herein that both of these changes decrease the rate of nucleation and the rate of growth of the fibril and increase the solubility of the amyloid fibril. To separately measure the effects of the two amino acid differences between the human and cat IAPP(20-29) sequences, two peptides, in which each change was introduced separately (IAPPH(20-29)F23L and IAPPH(20-29)S29P, Figure l ) , were studied, in addition to peptides based on the human (IAPPH(20-29)) and cat (IAPPC(20-29)) sequences.* These four peptides constitute a cycle, the characteristics of which provide information regarding the structural basis of amyloid formation. If residues 23 and 29 do not interact in the fibril, then the kinetic and thermodynamic effects of each individual 'mutation" would beexpected toadditive (ln(F23L) ln(S29P) = ln(overal1 effect), Figure l).9J0 In such a case, the effects of each "mutation" would
be context-independent (i.e,, S29P = S29P' and F23L = F23L') and the cycle would be rectangular. Alternatively, if residues 23 and 29 do interact, then the individual effects would be nonadditive (ln(F23L) ln(S29P) # ln(overal1 effect)) and contextdependent (e.g., S29P # S29P').lO Each peptide slowly formed amyloid fibrils (EM) from a supersaturated solution.11 The resultant fibrils produced indistinguishable FTIR spectra, suggestive of antiparallel @-sheet structure (maximumat ca. 1 6 3 0 ~ m - ~ ) Peptidefilms$formed .~~J~ by rapid evaporation of formic acid solutions, produced FTIR spectra similar to those of the fibrils, with one exception: the IAPF(20-29) film FTIR spectrum indicated disordered structure (broad absorption band centered at 1641 cm-1). This result suggested that amyloid formation was slowest for the cat sequence and inspired the kinetic studies discussed below. Amyloid formation is a nucleation-dependent process.l3-*6 The requirement for nucleation leads to a delay in the appearance of insoluble amyloid fibrils, or lag time (Figure 2). The duration of the lag time depends, in part, on the association equilibria leading to nucleus formation (&).I7 Each single amino acid change resulted in a ca. 3-fold increase in the lag time relative to IAPPH(20-29) (F23L = 2.7-fold, S29P = 3.6-fold; Table I), while the IAPPC(20-29) peptide nucleated approximately 13-
(1) Nishi, M.;Sanke, T.; Nagamatsu, S.; Bell, G. I.; Steiner, D. F. J. Biol. Chem. 1990, 265, 4173. (2) Glenner, G. G.; Eanes, D.;Wiley, C. Biochem. Biophys. Res. Commun. 1988, 155, 608. (3) Westermark, P.; Engstrom, U.; Johnson, K. H.; Westermark, G. T.; Betsholtz. C. Proc. Natl. Acad. Sci. U.S.A. 1990. 87. 5036. (4) Ashburn, T. T.; Auger, M.; Lansbury, P. T.,Jr. J . Am. Chem. Soc. 1992. 114. 790. One letter amino acid codes: A = Ala, C = Cys, F = Phe, G = Gly, H = His, I = Ile, K = Lys, N = Asn, P = Pro, Q = Gln, R = Arg, S = Ser, T = Thr, V = Val, Y = Tyr. (6) Rats do not develop pancreatic amyloid or type I1 diabetes.21 IAPPR(20-29) (AcHN-SNNLGPVLPP-CONH,) does not form amyloid fibrils (solubility = 60 500 k 3800 pM). Cats develop pancreatic amyloid in conjunction with type I1 diabetes.' (7) Betsholtz, C.; Christmanson, L.; Engstrom, U.;Rorsman, F.; Jordan, K.; O'Brien, T. D.; Murtaugh, M.; Johnson, K. H.; Westermark, P. Diabetes 1990, 39, 118. (8) Syntheses were performed using 9-fluorenylmethoxycarbnyl (Fmoc) amine protection and benzotriazol-l-yl-oxytris(pyrro1idino)phosphonium hexafluorophosphate (PyBOP) couplings. The Rink amide ((4-(2',4'dimethoxyphenyl)( Fmoc)aminomethylphenyl)oxy)acetamidonorleucyl-MBHA resin (Novabiochem) provided the C-terminal amide, and the N-terminus was acetylated with acetic anhydride. Serine (tert-butyl) and asparagine (trityl) were deprotected and the peptides cleaved from the resin according to a published procedure.22 Peptides were purified by isocratic reversed-phase high-pressure liquid chromatography (RPHPLC) on a YMC C18 column (300-A pore size, 15-pm particle size, 30 X 300 mm2). Purity of each peptide was judged to be >98% by isocratic analytical RPHPLC (Waters C4 column (300-.&, 3.9 X 30 "2)). Each peptide had an amino acid composition and plasma desorption mass spectrum consistent with the proposed primary structure.
(9) Perry, K. M.; Onuffer, J. J.; Gittelman, M. S.; Barmat, L.; Matthews, C. R. Biochemistry 1989, 28, 7961. (10) Ackers, G. K.; Smith, F. R. Annu. Rev. Biochem. 1985,54, 597. (1 1) Peptide aggregation was initiated by adding a solution'of the peptide (100 s L , 6.9 mM in DMSO by amino acid analysis (aaa)) to 900 pL of pH 7.4 buffer (100 mM NaCI, 1.8 mM NaH2P04, 8.2 mM NaZHPO,, 0.2% NaNp) in a 10- X 75-mm2disposableculturetube at room temperature. Samples werestirredat 1550rpmwitha3- X 10-mmzmagneticstirringbar. Turbidity was measured at 400 nm vs buffer. Thermodynamic solubility was determined after the aggregated solutions were stirred for 1 week. The samples were then centrifuged for25 minat 2100g, thesupernatants were filtered through MillexGV 0.22-pm aqueous filters (Millipore), and peptide concentrations were determined by aaa. (12) Krimm, S.; Bandekar, R. J.; Adu. Prof. Chem. 1986, 38, 181. (13) Jarrett, J. T.; Lansbury, P. T., Jr. Biochemistry 1992, 31, 12345. (14) Come, J. H.; Fraser, P. E.; Lansbury, P. T., Jr. Proc. Narl. Acad. Sci. U.S.A. 1993, 90, 5959. (15) Jarrett, J. T.; Berger, E. P.; Lansbury, P. T., Jr. Biochemistry 1993, 32, 4693. (16) Jarrett, J. T.; Lansbury, P. T., Jr. Cell 1993, 73, 1. (1 7) For the simplest aggregation mechanism, where the nucleus is assembled by successive addition of monomers, the lag time is exponentially dependent on the number of monomers required to form the nucleus.16 The aggregation experiments reported herein were done with consistent stirring, due to the need to suspend the aggregate to accurately measure turbidity." We have observed that stirring greatly increases the nucleation rate. In the case of IAPPH(2&29), the reaction order in peptide was determined to be between 1 and 2. In an unstirred case, where the reaction order could be much greater, small changes in K. (which may correspond to similar changes in K,) will have dramatic effects on the lag t i n ~ e . ~Stirring ~ J ~ may change the mechanism of amyloid formation by increasing the diffusion of small oligomers or by breaking up small fibrils to increase the number of growth faces.
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~
6)
0002-7863/93/1515-11012$04.00/0
ACHN-SNNLGAILSP-CONH, IAPPc(20-29)
Figure 1. Primary structure of IAPPH and the cycle created by the four peptides discussed in this paper.I0
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0 1993 American Chemical Society
Communications to the Editor
J . Am. Chem. SOC., Vol. 115, No. 23, 1993 11013
Table I peptide
lag time' (factorial increase relative to IAPPH(20-29)), s
thermodynamic solubilityb (factorial increase), LIM
growth rate X lo4 (factorial decrease). A 0 . D . d
IAPPH(2&29) seeded lag time! s
IAPPH(20-29) IAPPH(20-29)F23L IAPPH(20-29)S29P IAPPC(20-29)
310 840 (2.7) 1100 (3.6) 4000 (1 3)
9.0 14 (1.6) 32 (3.6) 109 (12)
25 5.8 (4.3) 4.2 (6.0) 1.O (25)
26 127 126 171
' Lag time observed for each peptide a t 690 pM. Each value is an average of a t least three separate experiments and was determined by solving for the best fit line to the growth phase of the aggregation curve for y = 0. The error in these measurements was
