Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5738-5742, August 1989 Biochemistry Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone (pancreatic beta celi/insulin/RIN-mSF insulinoma cells/calcitonin-gene-related peptide/polymerase chain reaction) MASAHIRO NISHI*, SHU JIN CHAN*t, SHINYA NAGAMATSU*, GRAEME 1. BELL*t, AND DONALD F. STEINER*t *Department of Biochemistry and Molecular Biology and tHoward Hughes Medical Institute, University of Chicago, Chicago, IL 60637 Contributed by Donald F. Steiner, May 3, 1989 ABSTRACT Islet amyloid polypeptide (IAPP) is a 37- precursor from cats, rats, mice, and guinea pigs and have amino acid peptide found in the pancreatic amyloid deposits of compared their deduced amino acid sequences to that of the type II (non-insulin-dependent) diabetic patients and insulino- human IAPP precursor (11). The results are consistent with mas. We previously reported the nucleotide sequence of a the view that IAPP (or its C-terminally amidated form) is a human cDNA, which indicated that IAPP is a C-terminally peptide hormone of the islets of Langerhans.A amidated peptide derived by proteolytic processing of an 89-amino acid precursor. We now report the isolation ofcDNA clones coding for cat, rat, mouse, and guinea pig IAPP MATERIALS AND METHODS precursors, obtained using the combination of "amplification Materials. Oligonucleotides were synthesized on an Ap- of homologous DNA fragments" (AHF) and "rapid amplifi- plied Biosystems model 380B DNA synthesizer. Genomic cation of cDNA ends" (RACE). The predicted structure of DNA was prepared from human peripheral blood lympho- IAPP precursors from these four mammals revealed that the cytes, rat liver (Sprague-Dawley), mouse kidney (BALB/c), LIPP moiety of each is derived from an 89- to 93-amino acid guinea pig (Hartley strain), and cat spleen as described (12). precursor by proteolytic processing and is likely to be amidated RNA was isolated from a rat insulinoma cell line (RIN-m5F; at the C terminus. The predicted amino acid sequence identities ref. 13) and mouse, guinea pig, and cat pancreas by the between the IAPP domains of these four m amm and human guanidinium thiocyanate/CsCl procedure (14). IAPP were 89% (cat), 84% (rat and mouse), and 78% (guinea Amplification of Homologous DNA Fragments (AHF) Par- pig). Within the IAPP domains, the N-terminal and C-terminal tially Encoding IAPP from Genomic DNA. A 119-base-pair amino acid sequences are very highly conserved among the (bp) DNA fragment coding for mature IAPP was amplified mammals, as is also the case with a structurally related from genomic DNA by the polymerase chain reaction (PCR) neuropeptide, calcitonin-gene-related peptide (CGRP), sug- using oligonucleotide primers A and B (Table 1). Each gesting that IAPP and CGRP interact with similar though not reaction mixture (100 A.l) contained 1 pug of DNA, 100 pmol identical receptors. By contrast, the N- and C-terminal propep- of each primer, and 2.5 units of Thermus aquaticus (Taq) tides of the IAPP precursor show very little sequence conser- DNA polymerase in 50 mM KCI/10 mM Tris Cl, pH 8.3/1.5 vation, which suggests that these regions do not represent mM MgCl2/0.01% (wt/vol) gelatin/200 .uM each deoxyribo- additional biologically active molecules. Interspecies variations nucleoside triphosphate (15). The PCR was carried out in a in the amino acid sequence of residues 20-29 of IAPP may Perkin-Elmer Cetus DNA thermal cycler through 30 cycles: account for the presence of amyloid deposits in the islets of denaturation for 1 min at 940C; annealing for 2 min at 50'C; humans and cats and their absence in rats and mice. and extension for 3 min at 650C. After extraction with chloroform, 30 A.l of the reaction mixture was electropho- A peptide structurally related to calcitonin-gene-related pep- resed in 7% polyacrylamide or 4% NuSieve GTG (FMC tide (CGRP) has been identified as a major protein compo- BioProducts) agarose gel. Amplified fragments of the ex- nent of amyloid deposits in the islets of Langerhans of pected size were eluted from the gel, subcloned into the diabetic humans and cats and in human insulinomas (1-5). HincII site of M13mpl8, and sequenced by the dideoxynu- Immunohistocytochemical studies (2, 6, 7) have identified the cleotide chain-termination procedure (16). beta cells of the islets as the source of this islet amyloid Rapid Amplification of cDNA Ends (RACE). The RACE polypeptide [IAPP; designated diabetes-associated peptide protocol for obtaining cDNA clones extended at the 3' or 5' (DAP) or amylin by some investigators]. Amyloid deposits ends was carried out essentially as described by Frohman et also occur in the islets of aging normal individuals and in al. (17) with minor modifications. cDNAs were prepared some insulinomas (8); however, they are more extensive in from 10 ug of total RNA (RIN-m5F cells, mouse and cat islets of type II diabetics, suggesting a possible role in the pancreas) or 0.5 ,ug of poly(A)+ RNA (guinea pig pancreas) pathogenesis ofthis disorder (4). Recent studies on the effects by using 200 fmol of oligo(dT)-adaptor as primer and avian of IAPP on the metabolism of glucose suggest that it selec- myeloblastosis virus reverse transcriptase. The reaction mix- tively inhibits insulin-stimulated glucose utilization and gly- tures were extracted with phenol/chloroform/isoamyl alco- cogen deposition in muscle, while not affecting adipocyte hol (25:24:1, vol/vol), precipitated with ethanol, and dis- glucose metabolism (9, 10). These observations have led to solved in 20 1.l of H20. The PCR for 3' RACE was then the hypothesis that IAPP may be a normal islet hormone that carried out on 1-4 A1l of the cDNA for 30 cycles (940C, 1 min; modulates the action of insulin on target tissues. If IAPP is a 50'C, 2 min; 65'C, 3 min) with specific sense-strand primers hormone of consequence, certain features of its structure (Table 2, 3'Amp). should be well conserved among various species. To examine this question, we have sequenced cDNAs encoding the IAPP Abbreviations: IAPP, islet amyloid polypeptide; CGRP, calcitonin- gene-related peptide; PCR, polymerase chain reaction; AHF, am- plification of homologous DNA fragments; RACE, rapid amplifica- The publication costs of this article were defrayed in part by page charge tion of cDNA ends. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M25387-M25390). 5738 Downloaded by guest on September 28, 2021 Biochemistry: Nishi et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5739 Table 1. Primers used for AHF PCR was reduced to 50°C. Although the resulting reaction Name Sequence mixtures also contain artifactual DNA fragments, since the 1 2 3 4 5 6 7 8 exact size of the target DNA is known, it can be readily Lys Cys Asn Thr Ala Thr Cys Ala purified from the contaminating fragments by electrophoresis Primer A 5' AAG TGC AAC ACT GCC ACA TGY GC in polyacrylamide or agarose gels (S.J.C., unpublished 34 35 36 37 38 39 40 Ser Asn Thr Tyr Gly Lys Arg results). This observation suggested to us that oligonucleo- Primer B 3' AGG TTA TGT ATA CCG TTC GC tide primers corresponding to a conserved region in the Oligo(dT)-adaptor 5' GAC TCG AGT CGA CAT CGA T17 human IAPP protein could be used to obtain partial IAPP Adaptor 5' GAC TCG AGT CGA CAT CG coding sequences from other species. Exon 3 ofthe human IAPP gene encodes amino acids 28-89 Amino acid numbering is relative to the N terminus of mature of the IAPP precursor and includes the IAPP moiety (T. IAPP. Sanke, M.N., and D.F.S., unpublished results). Since the cDNAs for 5' RACE were prepared by substituting specific exon/intron organization of other polypeptide hormone antisense primers for the oligo(dT)-adaptor in the reverse genes has been generally well conserved during evolution, we transcription reaction (Table 2, 5'RT). The cDNA was pre- tested primers A and B (Table 1) to determine whether they cipitated twice in 2.5 M ammonium acetate with 3 vol of could be used to amplify the region coding for IAPP in DNA ethanol and then tailed with oligo(dA) by using dATP and a from other animals. As shown in Fig. 1, a 119-bp DNA kit from Boehringer Mannheim. Double-stranded cDNA was fragment representing amino acids 1-37 of IAPP and the synthesized by the Klenow fragment of DNA polymerase I C-terminal Gly-Lys as well as two nucleotides of the follow- with oligo(dT)-adaptor (25 pmol) as primer at 15°C for 60 min. ing arginine codon was the major product in the PCR using The PCR was carried out with specific (internal) antisense human, cat, rat, mouse, or guinea pig DNA. The sequence of primers (Table 2, 5'Amp). The 3'- and 5'-RACE reaction the human fragment was identical to that of the correspond- mixtures were extracted with chloroform and the amplified ing region of the human gene. Similarly, we sequenced the cDNAs were subcloned into the HincHI site of pGEM-4Z. 119-bp fragments from the other species and these were Plasmids with IAPP cDNA inserts were identified by colony clearly homologous to the human sequence and represented hybridization (18) with 32P-labeled primer A or B and se- a segment of the IAPP gene of each of these animals.