Structure of Cat Islet Amyloid Polypeptide and Identification of Amino Acid Residues of Potential Significance for Islet Amyloid Formation

Abstract

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 chain-reaction 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.

Full text

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 chain-
reaction 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-de-
pendent (type II) diabetes mellitus in humans and
cats (1-5). The amyloid is composed predomi-
nantly 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 Min-
nesota, St. Paul, Minnesota; and Department of Pathology, University Hospital,
Linkoping,
Sweden.
Address correspondence and reprint requests to Christer Betsholtz, De-
partment of Pathology, University Hospital, S-751 85 Uppsala, Sweden.
Received for publication 14 August 1989 and accepted in revised form 14
September 1989.
pro-IAPP immunoreactivity has also been recently demon-
strated 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 pre-
cursor 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 patho-
genesis 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 (2,3,17-
19).
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
func-
tion 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, mon-
keys,
and cats develop islet amyloid-type II diabetes syn-
dromes that are similar regarding age dependency and
spontaneous onset (1-5,23). In contrast, most rodent spe-
cies 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 in-
trinsic 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 sub-
stantially 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-
118DIABETES, VOL. 39, JANUARY 1990

C. BETSHOLTZ AND ASSOCIATES
H
5 UT•f signal seq. | N-term.pp|3 UT
I A PP\ C-term. pp \-
B
Hamplified fragment
168 bp
I
gel 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. Consis-
tent with the high incidence of islet amyloid formation ob-
served in this species, amino acid region 20-29 was similar
in structure to the corresponding human region, and a cat
IAPP
20
_
29
synthetic peptide formed amyloidlike fibrils in vitro.
A cat/hamster IAPP20_29 hybrid peptide also formed amy-
loidlike 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'GCAAGCTTAGTCAT-
CAGGTGGAAAAGCG and 5'CGGAATTCTCTACTGCATTC-
CTCTTGC, were synthesized with Applied Biosystems
equipment (Forster City, CA). Amplification products of ex-
pected 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 Bio-
systems model 430A peptide synthesizer. The peptides were
purified by reverse-phase high-performance liquid chro-
matography 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 po-
larization microscopy. Small droplets were also applied to
formvar-coated copper grids, negatively contrasted with ur-
anyl acetate, and studied in a Jeol 100-SX electron micro-
scope at 80 kV.
Lys(l)Tyr(37)
cat
human
hamster
rat
mouse
AAATGCA
G
G
G
ACACTGCC
G
G
G
:ACATG:
G
G
G
rGCGA
A
C
C
C
CCC
G
A
A
A
AACGCCTC
G
GT
}GCAAATT
C
C
C
C
TCT
T
T
T
TAA1
G
GG
GG
GG
fTCGTTCCAGCAA
A
C
A G A
C
CAATCTTC
CT
C
C
C
}GTGC
C
C
C
;CAI
G
AG
AG
rTC
c
c
c
TTTC
C
C
CC
CC
;TCC
AT
A
A
A
3TACC
A
A
A
;AATGTGG
C
C
C
GATCCA
G
G
ATA
C
C
CATAT
G C
C
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.
DIABETES, VOL. 39, JANUARY 1990119

CAT ISLET AMYLOID POLYPEPTIDE
1015
20253035
K
K
K
K
K
C
C
C
C
C
N
N
I
N
N
T
T
T
T
T
A
A
A
A
A
T
T
T
T
T
C
C
C
C
C
A
A
A
A
A
T
T
T
T
T
Q
Q
Q
Q
Q
R
R
R
R
R
L
L
L
L
L
A
A
A
A
A
N
N
N
N
N
F
F
F
F
F
L
L
L
L
L
I
V
V
V
V
R
H
H
R
R
S
S
S
S
S
s
s
N
S
S
N
N
N
N
N
N
N
N
1ST
N
L
F
L
L
L
G
G
G
G
G
A
A
P
P
P
I
I
V
V
V
L
L
L
L
L
S
S
S
P
P
P
S
P
P
P
T
T
T
T
T
N
I
N
N
N
V
V
V
V
V
G
G
G
G
G
S
S
S
S
s
N
N
N
N
N
T
T
T
T
T
Y
Y
Y
Y
Y
cat
human
hamster
rat
mouse
FIG.
3.
Comparison
of
amino acid sequences
of
different islet amyloid poiypeptides.
-NH*
-NH2
-NH*
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 am-
plification (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 se-
quenced.
The resulting
cat
IAPP cDNA sequence
is
shown
in comparison with the human, hamster, rat, and mouse se-
quences (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 amy-
loid 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 micros-
copy 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
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 di-
abetes remain essentially unanswered: What is the cause of
amyloid formation? What role does amyloid play in the patho-
genesis of type II diabetes? This study provides an important
clue regarding the differences in islet amyloid formation be-
tween certain mammalian species. However,
the
conclu-
sions obtained obviously depend
on
whether
the in
vitro
amyloidogenic properties
of
the respective synthetic deca-
peptide studied (corresponding to amino acid residues 20-
29
of
the mature IAPP molecule) truly reflect the amyloido-
genic properties
of
the intact 37-amino acid IAPP molecule
or
its
precursor
in
vivo. The structure
of
IAPP has been de-
termined either
by
direct chemical analysis
or
cDNA
anal-
yses
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, espe-
cially 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
indi-
viduals.
The
human IAPP structure, deduced through
the
sequencing
of
cDNA and genomic DNA clones from
indi-
viduals apparently without islet amyloid deposits (12,13,30,
unpublished data),
is
identical to that obtained by sequenc-
ing 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
SpeciesSequenceFibrillogenesis Refs.
Human SNNFGAILSS
Yes
13,24,25
Cat
S N N L
G
A I L
S P
Yes
This study
Hamster NNNLGPVLSP
No 25
Cat/hamster NNNLGAI
LSP Yes
Tnis study
120DIABETES, VOL. 39, JANUARY 1990

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 1990121

CAT ISLET AMYLOID POLYPEPTIDE
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 Med-
ical 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 38:49-
59,
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 di-
abetes 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 pres-
ent 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 con-
tains 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 immunoreac-
tivity 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 pro-
cessing.
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
13.
Betsholtz C, Svensson V, Rorsman F, Engstrom U, Westermark GT, Wil-
ander 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: Co-
localization of islet amyloid polypeptide (IAPP) and insulin in the
B-cell
secretory granules of the human pancreatic islets. Diabetologia 32:240-
44,1989
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 individ-
uals.
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 amy-
loidosis. 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,
John-
son 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
anal-
ysis 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 spectrom-
etry. 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
122DIABETES, VOL. 39, JANUARY 1990