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Rapid Publications Structure of Cat Islet Amyloid Polypeptide and Identification of Ami no Acid Residues of Potential Significance for Islet Amyloid Formation CHRISTER BETSHOLTZ, LARS CHRISTMANSON, ULLA ENGSTROM, FREDRIK RORSMAN, KATHY JORDAN, TIMOTHY D. O'BRIEN, MICHAEL MURTAUGH, KENNETH H. JOHNSON, AND PER WESTERMARK

Cats and humans, unlike most rodent species, develop amyloid in the islets of Langerhans in conjunction with non-insulin-dependent diabetes mellitus. The amyloid consists of a 37-amino acid polypeptide referred to as islet amyloid polypeptide (IAPP). The primary structures of IAPP from human and three rodent species have previously been determined. Sequence divergence was seen in the region corresponding to amino acid residues 20-29, which in human IAPP has been suggested to confer the amyloidogenic properties to the molecule. Using polymerase chainreaction methodology, we determined the primary sequence of cat IAPP. Amino acid region 20-29 shows specific similarities and differences compared with human and rodent IAPP, respectively. A synthetic cat IAPP20_29 decapeptide formed amyloid fibrils spontaneously in vitro. Comparison between the structure and amyloid fibril-forming activity of various synthetic peptides suggests that the amino acid residues at positions 25-26 in mature IAPP are important for the amyloidogenic properties of the molecule. Diabetes 39:118-22, 1990

A

myloid deposits in the islets of Langerhans are a typical finding in association with non-insulin-dependent (type II) diabetes mellitus in humans and cats (1-5). The amyloid is composed predominantly of islet amyloid polypeptide (IAPP; also referred to as amylin or diabetes-associated peptide), a 37-amino acid polypeptide showing partial sequence identity with members of the calcitonin gene-related peptide (CGRP; 6-9), but From the Department of Pathology, University Hospital, and Ludwig Institute for Cancer Research, Uppsala Branch, Uppsala, Sweden; Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota; and Department of Pathology, University Hospital, Linkoping, Sweden. Address correspondence and reprint requests to Christer Betsholtz, Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden. Received for publication 14 August 1989 and accepted in revised form 14 September 1989.

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pro-IAPP immunoreactivity has also been recently demonstrated in human islet amyloid deposits (10). cDNA cloning of human IAPP has indicated that the 37-amino acid peptide is a normal proteolytic product of an 89-amino acid precursor that is amidated at the COOH-terminal (11-13). Its normal physiological function is not known, but its homology with the CGRPs, its colocalization with insulin in islet (3-cell secretory granules (14,15), and its proposed function as an inhibitor of insulin-stimulated uptake of glucose in skeletal muscle cells (16) all point to a role as a hormone. The significance of islet amyloid formation in the pathogenesis of type II diabetes remains controversial, due at least in part to the fact that islet amyloid also occurs in conjunction with increasing age in nondiabetic humans and cats (1-3,5). However, islet amyloid deposits in nondiabetic individuals are not as extensive as those of diabetic individuals (2,3,1719). Localization of amyloid in very close contact with the p-cells, interposed between p-cells and islet capillaries (20,21), suggests that some of the aberrations in p-cell function observed in type II diabetes are caused by the amyloid deposits per se (22). Also of interest is the species-specific occurrence of the islet amyloid-diabetes mellitus syndrome. Humans, monkeys, and cats develop islet amyloid-type II diabetes syndromes that are similar regarding age dependency and spontaneous onset (1-5,23). In contrast, most rodent species and dogs (which are known to produce IAPP) do not develop islet amyloid and also do not develop characteristic type II diabetic syndromes (14). A region corresponding to amino acid residues 20-29 of the human IAPP molecule has been shown to have an intrinsic capacity of forming amyloid fibrils in vitro (13,24). We recently showed that the IAPP molecules of three rodent species (which do not develop islet amyloid) diverge substantially from the human sequence in this region (25). We also showed that a synthetic peptide corresponding to amino acid region 20-29 of hamster IAPP lacked the ability to form amyloid fibrils in vitro (25). In this study, with polymerase chain-reaction (PCR) meth-

DIABETES, VOL. 39, JANUARY 1990

C. BETSHOLTZ AND ASSOCIATES

H

5 UT

3 UT •f signal seq. | N-term.pp|

I A PP

\ C-term. pp \-

B H

I gel

amplified fragment 168 bp

purification

i Hind IM/EcoRI digestion

I subcloning into M13 vector

1 sequencing FIG. 1. Principle for polymerase chain-reaction (PCR)-mediated islet amyloid polypeptide (lAPP) partial cDNA cloning. A: schematic map of human lAPP mRNA (cDNA) and approximate annealing sites for PCR primers. Restriction recognition-site sequence is linked to each primer. UT, untranslated sequence; signal seq., signal sequence coding sequence; N-term. pp, NH2-terminal propeptide-coding sequence; lAPP, lAPP-coding sequence; C-term. pp, COOH-terminal propeptide-coding sequence; H, H/ndlll site; E, EcoRI site. B: processing procedure for amplified fragment, bp, Base pairs.

odology, we determined the cat lAPP sequence. Consistent with the high incidence of islet amyloid formation observed in this species, amino acid region 20-29 was similar in structure to the corresponding human region, and a cat IAPP20_29 synthetic peptide formed amyloidlike fibrils in vitro. A cat/hamster IAPP20_29 hybrid peptide also formed amyloidlike fibrils in vitro, suggesting that the amyloidogenic properties of lAPP are dependent on the amino acid residues at positions 25 and 26.

RESEARCH DESIGN AND METHODS

Cat lAPP cDNA cloning by PCR. Cat pancreas total cellular RNA was prepared, converted to single-stranded cDNA, and used as template for enzymatic DNA amplification by PCR (26) as described previously (25). The two oligonucleotide primers employed in the reaction, 5'GCAAGCTTAGTCATCAGGTGGAAAAGCG and 5'CGGAATTCTCTACTGCATTCCTCTTGC, were synthesized with Applied Biosystems

equipment (Forster City, CA). Amplification products of expected size were isolated, subcloned into M13 derivatives, and sequenced as described previously (27). In vitro test for amyloid fibril-forming activity of synthetic lAPPs. This was performed as described previously (13). Briefly, decapeptides corresponding to amino acid residues 20-29 of cat lAPP and cat/hamster lAPP were synthesized by automated solid-phase technique on an Applied Biosystems model 430A peptide synthesizer. The peptides were purified by reverse-phase high-performance liquid chromatography and analyzed by mass spectrometry (28). The peptides were solubilized (5 mg/ml) in 10% NH4OH, and 50% acetic acid was slowly added to the clear solution until a gel was formed. Aliquots of this material were dried on glass slides, stained with Congo red, and analyzed by polarization microscopy. Small droplets were also applied to formvar-coated copper grids, negatively contrasted with uranyl acetate, and studied in a Jeol 100-SX electron microscope at 80 kV.

Lys(l) cat human hamster rat mouse

Tyr(37)

AAATGCA ACACTGCC:ACATG:rGCGACCCAACGCCTC}GCAAATTTCTTAA1fTCGTTCCAGCAACAATCTTC}GTGC;CAIrTCTTTC;TCC3TACC;AATGTGGGATCCA ATACATAT A G G T G A CT C AT C G C G C G G G C C T GG C A G A C A G C C C Gc C A A C GG G G G C A T C C C AG c CC A A C G C C G G G C A C T GG C C AG c CC A A

FIG. 2. Comparison of cat islet amyloid polypeptide (lAPP) cDNA sequence (corresponding to lAPP-coding region only) with lAPP cONA sequences previously determined. NH2-terminal Lys codon (position 1) and COOH-terminal Tyr codon (position 37) are indicated.


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CAT ISLET AMYLOID POLYPEPTIDE

10

25

20

15

30

35

cat

KC N T A T C A T Q R L A N F L I R S

s

N N L G A I L S P T N V G S N T Y -NH*

human

K C N T A T C A T Q R L A N F L V H S

s

N N F G A I L S S T I V G S N T Y -NH2

hamster

K C I T A T C A T Q R L A N F L V H S N N N L G P V L S P T N VG S N T Y

rat

K C N T A T C A T Q R L A N F L V R S S N 1ST L G P V L P P T N V G S N T Y

mouse

K C N T A T C A T Q R L A N F L V R S S N N L G P V L P P T N V G

s

N T Y -NH*

FIG. 3. Comparison of amino acid sequences of different islet amyloid poiypeptides.

RESULTS

Isolation of cat IAPP cDNA fragment by PCR. Due to the presumed low abundance of cat IAPP mRNA in the total pancreas mRNA preparation, we utilized PCR methodology instead of classic cDNA library construction and screening procedures. We have previously employed this technique to determine the IAPP sequence of three rodent species (25). The primers used were directed against the human IAPP cDNA sequence and were expected to be homologous enough with other mammalian IAPP sequences to allow amplification (11-13). To compensate for possible mismatches, we reduced the annealing temperature of the first two PCR cycles to 37°C (26). Figure 1 shows the principle for the amplification reaction. An amplification product of expected size (168 base pairs) was agarose gel purified, restriction enzyme digested, subcloned into M13 vectors, and sequenced. The resulting cat IAPP cDNA sequence is shown in comparison with the human, hamster, rat, and mouse sequences (Fig. 2). Specific sequence divergence in amyloidogenic region of IAPP. We compared the IAPP amino acid sequences from species studied to date (Fig. 3). A substantial variability is observed in amino acid region 17-29. The only positions that are consistently linked with the occurrence of islet amyloid formation are residues 25 and 26, i.e., Ala-lie in cat and human (which develop islet amyloid) and Pro-Val in hamster, rat, and mouse (which do not develop islet amyloid). In vitro amyloidogenic properties of synthetic amino acid regions 20-29. A synthetic peptide corresponding to cat IAPP amino acid residues 20-29 formed amyloidlike fibrils in vitro exhibiting green birefringence on Congo red staining and polarization microscopy (not shown). Electron microscopy showed that the fibrils were long and slightly curved and consisted of two or more parallel filaments of ~ 4 nm width (Fig. A A). The cat IAPP amino acid region 20-29 differs from the corresponding hamster region in three positions, 20, 25, and 26 (Fig. 3). To test the importance of positions 25 and 26 in fibrillogenesis, we synthesized a hamster IAPP20_29 peptide, with the Pro-Val sequence at positions 25 and 26 replaced by the respective residues (Ala-lie) found in human and cat IAPP. This cat/hamster hybrid peptide exerted fibrillogenic properties closely similar to those of the cat IAPP20_29 peptide (Fig. 4, B and C). Data obtained concerning the structure

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and in vitro fibrillogenic properties of different IAPP amino acid regions 20-29 are summarized in Table 1. DISCUSSION

Two central questions regarding islet amyloid in type II diabetes remain essentially unanswered: What is the cause of amyloid formation? What role does amyloid play in the pathogenesis of type II diabetes? This study provides an important clue regarding the differences in islet amyloid formation between certain mammalian species. However, the conclusions obtained obviously depend on whether the in vitro amyloidogenic properties of the respective synthetic decapeptide studied (corresponding to amino acid residues 2 0 29 of the mature IAPP molecule) truly reflect the amyloidogenic properties of the intact 37-amino acid IAPP molecule or its precursor in vivo. The structure of IAPP has been determined either by direct chemical analysis or cDNA analyses in five mammalian species (6-9,11-13,25,29, herein), and a correlation between the in vitro behavior of synthetic IAPP20-29 peptides and the observed in vivo incidence of islet amyloid formation has been demonstrated. The data point to specific differences in the IAPP primary structure, especially amino acid residues at positions 25 and 26, as potential determinants of the observed species differences. However, differences in primary sequence cannot explain quantitative differences in islet amyloid formation between human individuals. The human IAPP structure, deduced through the sequencing of cDNA and genomic DNA clones from individuals apparently without islet amyloid deposits (12,13,30, unpublished data), is identical to that obtained by sequencing IAPP isolated from islet or insulinoma amyloid (6-9). Thus, although a specific primary structure of IAPP may be

TABLE 1 Amino acid sequence and in vitro fibrillogenic properties of synthetic decapeptides corresponding to amino acid residues 20-29 of islet amyloid polypeptide Species

Sequence

Fibrillogenesis

Refs.

Human Cat Hamster Cat/hamster

SNNFGAILSS S NNLG A I LS P NNNLGPVLSP N N N L G A I LSP

Yes Yes No

13,24,25 This study 25 Tnis stud y

Yes


FIG. 4. Electron micrographs showing amyloidiike fibrils formed from synthetic decapeptides corresponding to islet amyloid polypeptide (IAPP) residues 20-29 (see Table 1 for peptide sequences). A: wide aggregates (up to 27 nm width) of filaments formed from cat IAPP20_29. B: similar aggregates formed from hamster/cat hybrid IAPP20-29- C: slender fibrils consisting of s2 filaments, each ~4 nm wide. Congophilia and green birefringence with polarized light (characteristic feature of amyloid) were observed when fibrils in A-C were exposed to Congo red dye. x 110,000. DIABETES, VOL. 39, JANUARY 1990

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a prerequisite for amyloidogenesis, other factors must also be involved in determining whether islet amyloid will form. These factors are not known, but may include aberrations in synthesis, processing, release, or paracellular clearance of IAPP (31). ACKNOWLEDGMENTS

This study was supported by grants from the Swedish Medical Research Council, the Research Fund of King Gustaf V, the Nordic Insulin Fund, the Louis-Hansen's Memorial Fund, and the National Institute of Diabetes and Digestive and Kidney Diseases. We thank Dr. Eva Seligsohn for providing cat pancreas biopsies. REFERENCES 1. Bell ET: Hyalinization of the islets of Langerhans in diabetes mellitus. Diabetes 1:341-44, 1952 2. Erlich JC, Ratner IM: Amyloidosis of the islets of Langerhans: a restudy of islet hyalin in diabetic and nondiabetic individuals. Am J Pathol 38:4959, 1961 3. Westermark P: Quantitative studies of amyloid in the islets of Langerhans. Upsala J Med Sci 77:91-94, 1972 4. Yano BL, Hayden DW, Johnson KH: Feline insular amyloid: association with diabetes mellitus. Vet Pathol 18:621-27, 1981 5. Johnson KH, Hayden DW, O'Brien TD, Westermark P: Spontaneous diabetes mellitus—islet amyloid complex in adult cats. Am J Pathol 125:4165-69, 1986 6. Westermark P, Wernstedt C, Wilander E, Sletten K: A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem Biophys Res Commun 140:827-31, 1936 7. Westermark P, Wernstedt C, Wilander E, Hayden DW, O'Brien TD, Johnson KH: Amyloid fibrils in human insulinoma and islets of Langerhans in the diabetic cat are derived from a novel neuropeptide-like protein also present in normal islet cells. Proc Natl Acad Sci USA 84:3881-85, 1987 8. Westermark P, Wernstedt C, O'Brien TD, Hayden DW, Johnson KH: Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. Am J Pathol 127:414-17, 1987 9. Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Reid KBM: Purification and characterization of a peptide from amyloid-rich pancreas of type 2 diabetic patients. Proc Natl Acad Sci USA 84:8628-32, 1987 10. Westermark P, Engstrom U, Westermark GT, Johnson KH, Permerth J, Betsholtz C: Islet amyloid polypeptide (IAPP) and pro-IAPP immunoreactivity in human islets of Langerhans. Diabetes Res Clin Pract 7:219-26, 1989 11. Sanke T, Bell Gl, Sample C, Rubenstein AH, Steiner DF: An islet amyloid peptide is derived from an 89-amino-acid precursor by proteolytic processing. J Biol Chem 263:17243-46, 1988 12. Mosselman S, Hoppener JWM, Lips CJM, Jansz HS: The complete islet amyloid polypeptide precursor is encoded by two exons. FEBS Lett 247:154-58, 1989

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13. Betsholtz C, Svensson V, Rorsman F, Engstrom U, Westermark GT, Wilander E, Johnson KH, Westermark P: Islet amyloid polypeptide (IAPP): cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus. Exp Cell Res 183:484-93, 1989 14. Johnson KH, O'Brien TD, Haten DW, Jordan K, Ghobrial HKG, Mahoney WC, Westermark P: Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidase antiperoxidase (PAP) and protein A-gold techniques. Am J Pathol 130:1-8, 1988 15. Lukinius A, Wilander E, Westermark GT, Engstrom U, Westermark P: Colocalization of islet amyloid polypeptide (IAPP) and insulin in the B-cell secretory granules of the human pancreatic islets. Diabetologia 32:24044,1989 16. Leigton B, Cooper GJS: Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature (Lond) 335:632-35, 1988 17. Bell ET: Hyalinization of the islets of Langerhans in nondiabetic individuals. Am J Pathol 35:801-805, 1959 18. Westermark P, Grimelius P: The pancreatic islet cells in insular amyloidosis in human diabetic and non-diabetic adults. Acta Pathol Microbiol Scand Sect A Pathol 81:291-300, 1973 19. Clark A, Cooper GJS, Lewis CE, Morris JF, Willis AC, Reid KBM: Islet amyloid formed from diabetes-associated peptide may be pathogenic in type 2 diabetes. Lancet 2:231-34, 1987 20. Westermark P: Fine structure of islets of Langerhans in insular amyloidosis. Virchows Arch Abt A Pathol Anat 38:49-59, 1973 21. Westermark P, Wilander E: The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus. Diabetologia 15:417-21,1978 22. Type 2 diabetes or NIDDM: looking for a better name (Editorial). Lance? 1:589-91, 1989 23. Howard CF Jr: Insular amyloidosis and diabetes mellitus in Macaca nigra. Diabetes 27:357-64, 1978 24. Glenner GG, Eanes ED, Wiley CA: Amyloid fibrils formed from a segment of the pancreatic islet amyloid protein. Biochem Biophys Res Commun 155:608-14, 1988 25. Betsholtz C, Christmanson L, Engstrom U, Rorsman F, Svensson V, Johnson KH, Westermark P: Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species. FEBS Lett 251:261-64, 1989 26. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-54, 1985 27. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-67, 1977 28. Sundqvist B, MacFarlane RD: 252Cf-plasma desorption mass spectrometry. Mass Spectro Rev 4:421-60, 1985 29. Leffert JD, Newgard CB, Okamoto H, Milburn JL, Luskey KL: Rat amylin: cloning and tissue-specific expression in pancreatic islets. Proc Natl Acad Sci USA 86:3127-30, 1939 30. Mosselman S, Hoppener JWM, Zandberg J, van Mansfeld ADM, Guerts van Kessel AHM, Lips CJM, Jansz HS: Islet amyloid polypeptide (IAPP): identification and chromosomal localization of the human gene. FEBS Lett 239:227-32, 1988 31. Johnson KH, O'Brien TD, Jordan K, Westermark P: Impaired glucose tolerance is associated with increased islet amyloid polypeptide (IAPP) immunoreactivity in pancreatic beta cells. Am J Pathol 135:245-50, 1989
