From the Department of Pathology and Laboratory Medicine and the British Columbia Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada
Received for publication, September 14, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Islet amyloid deposits are a
characteristic pathologic lesion of the pancreas in type 2 diabetes and
are composed primarily of the islet beta cell peptide islet amyloid
polypeptide (IAPP or amylin) as well as the basement membrane heparan
sulfate proteoglycan perlecan. Impaired processing of the IAPP
precursor has been implicated in the mechanism of islet amyloid
formation. The N- and C-terminal cleavage sites where pro-IAPP
is processed by prohormone convertases contain a series of basic
amino acid residues that we hypothesized may interact with heparan
sulfate proteoglycans. This possibility was tested using affinity
chromatography by applying synthetic fragments of pro-IAPP to
heparin-agarose and heparan sulfate-Sepharose. An N-terminal human
pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose
and heparan sulfate-Sepharose, eluting at 0.18 M NaCl
at pH 7.5. Substitution of alanine residues for two basic residues in
the N-terminal cleavage site abolished heparin and heparan sulfate
binding activity. At pH 5.5, the affinity of the wild-type peptide for
heparin/heparan sulfate was increased, implying a role for histidine
residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP
fragment (residues 41-67) had no specific affinity for either heparin
or heparan sulfate, and the N- or C-terminal fragments had only weak
affinity for chondroitin sulfate. These data suggest that monomeric
N-terminal human pro-IAPP contains a heparin binding domain that is
lost during normal processing of pro-IAPP.
Type 2 diabetes is characterized by peripheral insulin resistance
(1) coupled with a progressive loss of insulin secretion (2) that is
associated with a decrease in pancreatic islet beta cell mass and the
deposition of amyloid in the pancreatic islets (3, 4). The principal
component of islet amyloid is a 37-amino acid peptide called islet
amyloid polypeptide (IAPP1 or
amylin) (5, 6). IAPP is a normal product of the islet beta cell (7, 8)
and is cosecreted from beta cells along with insulin (9-12). In type 2 diabetes, IAPP aggregates to form amyloid fibrils within the islet that
are thought to be toxic to islet beta cells (13). Other components of
islet amyloid that are common to all amyloids include apolipoprotein E
(3, 14) and the heparan sulfate proteoglycan, perlecan (3, 15).
Heparan sulfate proteoglycans, and in particular the basement membrane
proteoglycan perlecan, have been proposed to play an important role in
amyloid deposition in Alzheimer's disease, familial amyloidoses, prion
diseases, and type 2 diabetes (16, 17). Heparan sulfate proteoglycans
are widely expressed and are an important component of extracellular
matrix and basement membranes, where amyloid tends to accumulate. Many
proteins, for example lipoprotein lipase (18) and apolipoprotein E
(19), interact with heparan sulfate proteoglycans via electrostatic
interaction of heparin binding domains (clusters of basic residues)
with the dense negative charge on the highly sulfated glycosaminoglycan chains. In many cases, these interactions serve important physiological roles, but interaction of amyloidogenic precursors with heparan sulfate
proteoglycans may also play an important initiating role in amyloid
formation (17). Most amyloidogenic precursor proteins, including the
Alzheimer's precursor protein (20, 21) and serum amyloid A (SAA) (22),
have been shown to have heparin binding domains that facilitate their
interaction with glycosaminoglycans. These heparin binding domains
usually, but not always, follow the consensus sequence for heparin
binding proposed by Cardin and Weintraub (23) as follows:
XBBBXXBX or
XBBXBX, where B is a basic amino acid
and X is a nonbasic amino acid. Although a heparin binding
domain in IAPP has not yet been identified, fibrils derived by in
vitro aggregation of synthetic human IAPP have been shown to bind
to both heparin (24) and perlecan (25).
Although the mechanism of islet amyloid formation is unknown, one
potential cause has been proposed to be alterations in the processing
of the IAPP precursor molecule, pro-IAPP, by the islet beta cell (3,
26). Pro-IAPP is processed to mature IAPP in beta cell secretory
granules by the action of prohormone convertase enzymes at the
C-terminal side of dibasic residues. The prohormone convertases PC2 and
PC3 (also called PC1) are likely responsible for pro-IAPP processing,
since they are present in beta cell granules, are known to similarly
process proinsulin at the C-terminal side of dibasic residues (27, 28),
and appear to be capable of fully processing pro-IAPP in
vitro (29). Furthermore, we have recently shown that PC2 is
essential for processing of pro-IAPP at its N-terminal cleavage site
(30). In type 2 diabetes, processing of proinsulin by beta cells is
defective, resulting in elevated release of proinsulin relative to
insulin (31, 32). Since proinsulin and pro-IAPP are processed in
parallel in beta cells, it seems likely that pro-IAPP processing may
also be defective in this disease, leading to the hypersecretion of
unprocessed or partially processed forms of pro-IAPP. Interestingly,
immunoreactivity for the N-terminal (but not the C-terminal) region of
pro-IAPP has been found to be present in islet amyloid deposits (33) suggesting that partially processed, N-terminally extended pro-IAPP might be an important molecule in islet amyloid formation.
We hypothesized that unprocessed or partially processed pro-IAPP might
interact with basement heparan sulfate proteoglycans. To test this
hypothesis, we synthesized fragments of pro-IAPP containing the N- and
C-terminal cleavage sites, and we assessed their ability to bind to
heparin and heparan sulfate.
Materials--
Heparin, heparan sulfate, chondroitin sulfate A,
bovine serum albumin (BSA), Sepharose CL-4B, heparin-agarose CL-4B, and
thioflavin T were purchased from Sigma. CNBr-activated Sepharose 4B was
purchased from Amersham Pharmacia Biotech. Wild-type and mutant
pro-IAPP fragments were synthesized at the Nucleic Acid Protein
Synthesis unit at the University of British Columbia. All synthetic
peptides were high performance liquid chromatography-purified, and the N-terminal peptides containing cysteine residues at positions 6 and 9 of IAPP were cyclized. Rat and human IAPP were purchased from Bachem
(Torrance, CA).
Affinity Chromatography--
Heparin-agarose affinity
chromatography was performed as per Ancsin and Kisilevsky (22). In
brief, an 8-ml column of heparin-agarose was equilibrated with 20 mM Tris-HCl at either pH 7.5 or 5.5, as indicated. Peptide
(300 µg) was dissolved in buffer and loaded onto the column, washed
with four column volumes of buffer, and then developed at a flow rate
of 0.5 ml/min with a 0-1 M NaCl gradient over five column
volumes using a GM-1 gradient mixer (Amersham Pharmacia Biotech).
Fractions (0.5 ml) were collected, and absorbance was measured at 214 nm. Salt concentrations in fractions were directly measured using a
hand-held conductivity meter. Heparan sulfate and chondroitin sulfate
affinity chromatography were performed in a similar manner, except the
columns were made by coupling free heparan sulfate or chondroitin
sulfate to CNBr-activated Sepharose 4B beads following the protocol
provided by the manufacturer. Glycosaminoglycan coupling efficiency to
the Sepharose beads, assessed by the toluidine blue method (34) was
found to be 0.25 mg/ml gel for chondroitin sulfate and 0.1 mg/ml gel
for heparan sulfate. To minimize cost for preparation of heparan
sulfate-Sepharose the coupling protocol was scaled down, and a 3-ml
column was used.
Fluorometry--
The degree of peptide aggregation to form
amyloid fibrils was determined by thioflavin T fluorescence using an
assay adapted from Naiki et al. (35) and Kudva et
al. (36) for a 96-well plate format. All peptides were dissolved
in 100% dimethyl sulfoxide (Me2SO), aliquoted, and
kept frozen as 250 µM stock solutions until thawing
immediately prior to use. Peptide was added to wells containing 10 µM thioflavin T in 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.1% Triton X-100 to a final peptide
concentration of 12.5 µM and final Me2SO
concentration of 5%. The plate was sealed with parafilm and
fluorescence measured at 37 °C using a Fluoroskan (Labsystems,
Vista, CA) fluorometer with filters set at 444 (excitation) and 485 (emission) nm and bandwidth slits of 12 and 14 nm, respectively. When
bound to amyloid fibrils, thioflavin T fluoresces with excitation and
emission maxima of 450 and 482 nm, respectively (35), and fluorescence
under these conditions correlates with the degree of amyloid fibril
formation as assessed by electron microscopy (36). Triplicate
measurements for each peptide were made every minute from 0 to 100 min,
every 10 min from 100 to 150 min, and finally every 60 min from 150 min
to 15 h, allowing kinetic assessment of IAPP fibrillogenesis. The
mean data were plotted and analyzed using KaleidagraphTM
software (Synergy Software, Reading, PA). Since amyloid fibril elongation is thought to follow first-order rate kinetics (37, 38), the
data were fit using nonlinear least squares to the equation:
Ft = F Affinity of Human Pro-IAPP Fragments for Heparin--
To test
whether the cluster of basic amino acids in the regions of the N- and
C-terminal cleavage sites in pro-IAPP might constitute heparin binding
domains, we synthesized peptides corresponding to the 30 N-terminal or
27 C-terminal amino acids of human pro-IAPP (see Fig.
1), containing these domains, and applied
these peptides to a heparin-agarose column. Since fibril formation
might enhance heparin binding (24), a critical amyloidogenic region in
IAPP (amino acids 31-40 of pro-IAPP) was omitted from these synthetic peptides to minimize the likelihood of protein aggregation. The N-terminal pro-IAPP fragment was retained by the heparin column, eluting at 0.18 M NaCl on a 0-1 M NaCl
gradient developed over 80 min (Fig.
2A). In contrast, BSA, a
protein known to not bind to heparin (22), eluted in the void volume.
When applied to a column containing uncoupled Sepharose, the N-terminal
pro-IAPP fragment eluted in the void volume (Fig. 2A),
indicating that its retention on heparin-agarose was not due to a
nonspecific interaction between the peptide and the column matrix. The
C-terminal region of pro-IAPP contains two pairs of basic residues,
including one at the C-terminal cleavage site involved in processing of pro-IAPP to IAPP-(1-37). Unlike the N-terminal pro-IAPP
fragment, the C-terminal pro-IAPP peptide interacted only weakly with
heparin, eluting in wash fractions prior to commencement of the NaCl
gradient (Fig. 2B).
To determine whether the
Lys10-Arg11-Lys12 sequence
in the N-terminal pro-IAPP cleavage site is essential for heparin
binding, we synthesized a peptide in which alanine residues were
substituted for the two basic residues (lysine and arginine) that
compose the cleavage site recognized by prohormone convertases during normal pro-IAPP processing (see Fig. 1). The K10A/R11A mutant N-terminal pro-IAPP peptide fragment had no affinity for heparin (Fig.
2C), eluting in the void volume when applied to the
heparin-agarose column. The complete loss of heparin binding in the
K10A/R11A mutant peptide indicates that one or both of the basic
residues in the N-terminal cleavage site of pro-IAPP are essential for heparin binding.
Affinity of Human Pro-IAPP Fragments for Heparan Sulfate--
To
determine whether the N-terminal region of human pro-IAPP has the
ability to bind to the heparan sulfate chains of heparan sulfate
proteoglycans, as it does heparin, we applied the synthetic N-terminal
pro-IAPP fragment to a column in which heparan sulfate was coupled to
Sepharose. As shown in Fig.
3A, the N-terminal pro-IAPP
peptide bound to the heparan sulfate column, eluting at a NaCl
concentration (0.17 M) almost identical to that observed when the peptide was applied to heparin-agarose. The C-terminal pro-IAPP fragment did not, by contrast, bind to the heparan sulfate, eluting in wash fractions (Fig. 3A). Similarly, the
K10A/R11A mutant N-terminal pro-IAPP fragment eluted in the void volume when applied to the heparan sulfate-Sepharose column (Fig.
3A). Thus, the cluster of basic residues in the N-terminal
cleavage site of pro-IAPP appears to confer affinity of this molecule
for both heparin and heparan sulfate.
To elucidate which of the basic residues in the N-terminal
cleavage site of pro-IAPP may be critical for heparan sulfate binding, we next synthesized two mutant peptides, in which an alanine residue was substituted for either the lysine at position 10 (K10A) or the
arginine at position 11 (R11A) of pro-IAPP. When applied to the heparan
sulfate column, both the K10A and the R11A mutant peptides eluted in
the void volume (Fig. 3B), indicating that both of these
basic residues are critical for interaction of N-terminal pro-IAPP with
heparan sulfate.
Affinity of Human Pro-IAPP Fragments for Chondroitin
Sulfate--
When applied to a chondroitin sulfate-Sepharose column,
both the N-terminal and C-terminal pro-IAPP peptide fragments appeared to have weak interaction with this glycosaminoglycan, eluting after the
void volume but prior to commencement of the salt gradient (Fig.
4). Thus, the affinity of the N-terminal
pro-IAPP peptide appears to be much stronger for heparan sulfate than
for chondroitin sulfate. Nonetheless, the weak binding of the
N-terminal pro-IAPP peptide to chondroitin sulfate does seem to be
dependent on the presence of the basic residues in the N-terminal
cleavage site, since the K10A/R11A mutant N-terminal pro-IAPP peptide
did not bind to chondroitin sulfate, eluting in the void volume (Fig. 4).
Human N- and C-terminal Pro-IAPP Fragments Are
Non-Fibrillogenic--
Amyloid fibrils formed by mature
human IAPP (pro-IAPP-(12-49)) and other amyloidogenic peptides
including the amyloid- Involvement of Histidine Residues in Heparin Binding at Acidic
pH--
By having determined that both the lysine and arginine
residues at positions 10 and 11 of N-terminal pro-IAPP are crucial for
its ability to bind heparan sulfate at pH 7.5 (Fig. 3B), we next investigated the involvement of histidine residues, which would be
protonated in the acidic milieu of beta cell secretory granules. There
are two histidine residues present in human pro-IAPP, one in the
N-terminal flanking region (position 6 of pro-IAPP) and one at amino
acid 18 of mature IAPP (position 29 of pro-IAPP) (see Fig. 1). At pH
5.5, the heparin affinity of the N-terminal human pro-IAPP fragment was
increased, with the peptide eluting at 0.28 M NaCl
(versus 0.18 M at pH 7.5) (Fig.
6A). The affinity of the
peptide for heparan sulfate was similarly increased at acidic pH (Fig.
6B). However, the K10A/R11A mutant had little affinity for
heparin (Fig. 6A) or heparan sulfate (Fig. 6B)
under these conditions. Thus, although either or both of the two
histidine residues present in pro-IAPP may increase its affinity for
heparin/heparan sulfate at acidic pH, they cannot substitute for the
Lys10-Arg11-Lys12 sequence in the
N-terminal cleavage site, which is still essential for heparin binding
at either acidic or neutral pH.
In addition to IAPP, islet amyloid deposits have been shown to
contain immunoreactivity for the N-terminal region of pro-IAPP (the
IAPP precursor) (33) and for basement membrane heparan sulfate
proteoglycan (15). The presence of these molecules in islet amyloid
deposits suggests their involvement in the mechanism of islet amyloid
deposition, yet it is unknown what role they might play. In the present
study, we demonstrate that a synthetic fragment of the N-terminal
region of pro-IAPP binds to heparin and heparan sulfate. We further
demonstrate that the heparin binding domain in N-terminally extended
pro-IAPP requires the presence of basic residues in the N-terminal
cleavage site at which one step in pro-IAPP processing occurs and,
therefore, that normal processing of pro-IAPP would be predicted to
destroy this heparin binding domain. The C-terminal region of pro-IAPP
showed no affinity for heparin or heparan sulfate, despite the presence
of two pairs of basic residues in this region. Our findings raise the
possibility that if secretion of unprocessed pro-IAPP (or partially
processed, N-terminally extended pro-IAPP) from the beta cell is
increased in type 2 diabetes, it might bind to the sulfated
glycosaminoglycan side chains of heparan sulfate proteoglycans,
creating a nidus for amyloidogenesis within the pancreatic islet. We
speculate that this mechanism may be an important initiating step in
islet amyloid formation in type 2 diabetes.
Pro-IAPP is thought to be processed to mature IAPP in beta cell
secretory granules by the action of PC2 and/or PC3 (PC1) cleaving on
the C-terminal side of pairs of basic residues (in both cases lysine-arginine), followed by trimming of these basic residues by
carboxypeptidase E (30). As a result, normal pro-IAPP processing results in the removal of the basic residues that we have shown are
critical for heparin binding of the N-terminal region of pro-IAPP. Interestingly, the
Lys10-Arg11-Lys12 cluster of basic
residues in the N-terminal pro-IAPP fragment does not represent a
classic linear heparin binding domain as proposed by Cardin and
Weintraub (23); however, many proteins that bind heparin do not possess
these sequences (39, 40). One model has suggested that a spacing of
~20 Å between two basic amino acids is a critical determinant of
heparin binding ability (41). Such spacing can be achieved by a peptide
in Heparin binding domains may also be created by protein aggregation, as
illustrated by the binding to heparin (24) and perlecan (25) of
aggregated human IAPP. Since our synthetic N- and C-terminal pro-IAPP
fragments remained soluble and did not form fibrils as assessed by
thioflavin T fluorescence, our findings cannot be explained by prior
aggregation of the N-terminal pro-IAPP peptide to form a heparin
binding domain. It is possible, however, that binding of soluble
pro-IAPP to heparin/heparan sulfate might induce conformational changes
in the protein that would enhance fibrillogenesis. Indeed, binding of
soluble amyloid- Our finding that heparin/heparan sulfate binding activity
of the N-terminal pro-IAPP peptide is increased at pH 5.5 raises the
possibility that alterations in the local pH, for example in acidic
intracellular compartments, might impact pro-IAPP binding to heparan
sulfate proteoglycans and subsequently amyloidogenesis. Alternatively,
it cannot be ruled out that such interactions might play a normal
physiological role, for example in (pro)IAPP trafficking and/or
processing, although it is unknown whether heparan sulfate proteoglycans are a component of the acidic beta cell secretory granules in which (pro)IAPP resides. Heparan sulfate proteoglycans have
been shown to be secreted via the constitutive pathway in other
secretory cells (43), and we have recently found that immature
(neonatal) rat beta cells secrete a significant proportion of (pro)IAPP
immunoreactivity by the constitutive secretory pathway (12). The
possible significance of the increased heparin binding of pro-IAPP at
acidic pH in both amyloid pathogenesis and in normal physiology may
therefore be worthy of further investigation.
The affinity of the N-terminal pro-IAPP peptide for another highly
sulfated glycosaminoglycan, chondroitin sulfate, was much less than
that observed for heparan sulfate. The weak interaction between
chondroitin sulfate and the N-terminal pro-IAPP peptide likely does,
however, involve the
Lys10-Arg11-Lys12 sequence in the
N-terminal cleavage site, since substitution of either the
Lys10 or Arg11 residues with alanines resulted
in total loss of binding. Chondroitin sulfate has not been shown to be
a component of islet amyloid, although it may be a component of amyloid
deposits in experimental murine inflammation-associated (AA)
amyloidosis (44) and Alzheimer's disease (45). This glycosaminoglycan
has also been shown to bind to human IAPP-derived fibrils in
vitro, albeit less strongly than does heparan sulfate (25). The
finding that the N-terminal pro-IAPP peptide binds to heparan sulfate
more avidly than it does to chondroitin sulfate, despite the higher
degree of sulfation of the latter, suggests that the degree of
sulfation is not the most important determinant of sulfated
glycosaminoglycan interaction with pro-IAPP. We speculate that the
spacings of the sulfate groups in heparan sulfate and heparin are more
appropriate than that of chondroitin sulfate for their interaction with
pro-IAPP, as has been recently suggested for interaction of
serum amyloid A, another amyloidogenic precursor, with
glycosaminoglycans (22).
Altered proteolytic processing of precursors to produce a
more amyloidogenic molecule may be a general mechanism underlying amyloid deposition in several different amyloidoses. This idea was
first suggested by the work of Glenner et al. (46) on
immunoglobulin light chain (AL) amyloidosis but has since been
implicated in the mechanism of amyloid formation in Alzheimer's
disease (17, 47, 48) and recently in familial British dementia (49). In
the case of islet amyloid, we propose that impaired proteolytic processing of pro-IAPP by beta cells may result in disproportionate secretion of forms of (pro)IAPP with high affinity for heparan sulfate
proteoglycans present on islet cell basement membranes. Once exocytosed
from the beta cell, these molecules may bind to perlecan on the
basement membranes of beta cells or islet vascular endothelial cells,
preventing their entry into islet capillaries. Indeed, ultrastructural
evidence from a transgenic mouse model of islet amyloid formation
suggests that amyloid fibrils first accumulate extracellularly between
islet beta cells and blood vessels (50). Interaction with the sulfated
glycosaminoglycan side chains might then induce conformational changes
in pro-IAPP that favor
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(F
F0)e
kt, where
Ft is the fluorescence at time t;
F
is the steady state fluorescence, and
k is the initial rate constant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence of full-length human
pro-IAPP (A) and the synthetic N- and C-terminal
pro-IAPP peptides used in these studies (B).
Arrows denote sites of pro-IAPP cleavage during normal
processing by prohormone convertases PC2 and PC3 to produce mature IAPP
(in bold). Boxed amino acids in A
represent sequence of synthetic peptides shown in B. + designates basic amino acid; underlined residues designate
alanine substitutions.
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Fig. 2.
Affinity of pro-IAPP fragments for
heparin. Synthetic peptides corresponding to human pro-IAPP amino
acids 1-30 (N-terminal fragment) (A), human
pro-IAPP-(41-67) (C-terminal fragment) (B), or human
pro-IAPP-(1-30) with alanines substituted for
Lys10-Arg11 (K10A/R11A) (C) were
applied to a heparin-agarose or uncoupled Sepharose column as indicated
in A. Bovine serum albumin (BSA) was used as a
control in A. The column was washed with four column volumes
of buffer (20 mM Tris-HCl, pH 7.5), developed with a 0-1
M NaCl gradient, fractions (0.5 ml) collected, and
absorbance determined at 214 nm.
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Fig. 3.
Affinity of pro-IAPP fragments for heparan
sulfate. Synthetic peptides corresponding to human pro-IAPP amino
acids 1-30 (N-terminal fragment), 41-67 (C-terminal fragment), or
1-30 with two alanine substitutions (K10A/R11A) (A) or
human pro-IAPP-(1-30) with single alanine substitutions (K10A or R11A)
(B) were applied to a heparan sulfate-Sepharose column. The
column was washed with four column volumes of buffer (20 mM
Tris-HCl, pH 7.5), developed with a 0-1 M NaCl gradient,
fractions (0.5 ml) collected, and absorbance determined at 214 nm.
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Fig. 4.
Affinity of pro-IAPP fragments
for chondroitin sulfate. Synthetic peptides corresponding to human
pro-IAPP amino acids 1-30 (N-terminal fragment), 41-67 (C-terminal
fragment), or 1-30 with alanine substitutions (K10A/R11A) were applied
to a chondroitin sulfate-Sepharose column. The column was washed with
four column volumes of buffer (20 mM Tris-HCl, pH 7.5),
developed with a 0-1 M NaCl gradient, fractions (0.5 ml)
collected, and absorbance determined at 214 nm.
protein of Alzheimer's disease are known to
bind to heparin (24). This affinity is thought to be dependent on the
aggregation state of the peptide, since human but not nonfibrillogenic
rodent IAPP has been found to bind to both heparin (24) and perlecan
(25). To rule out the possibility that the heparin binding activity of
our synthetic N-terminal pro-IAPP peptide was simply due to its
aggregation into fibrils that subsequently bound heparin, we measured
fibril formation using thioflavin T fluorescence. As expected,
thioflavin T fluorescence rapidly increased in the presence of human
IAPP but was unchanged in the presence of rat IAPP, demonstrating the
known fibrillogenic properties of human but not rat IAPP (Fig.
5). Neither the N- nor the C-terminal
pro-IAPP fragments formed fibrils as measured by thioflavin T
fluorescence (Fig. 5), producing identical traces to nonfibrillar rat
IAPP. These data indicate that both N- and C-terminal pro-IAPP
fragments were not fibrillar over the time course of these experiments
and therefore that the affinity for heparin and heparan sulfate
demonstrated by N-terminal pro-IAPP was not due to its prior
aggregation.
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Fig. 5.
N-terminal and C-terminal pro-IAPP fragments
are not fibrillogenic. Synthetic peptides (12.5 µM)
corresponding to human pro-IAPP amino acids 1-30 (N-terminal
fragment), human pro-IAPP amino acids 41-67 (C-terminal fragment),
mature human IAPP (pro-IAPP-(12-49)), or rat IAPP were incubated in 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Triton
X-100, and 10 µM thioflavin T. Amyloid fibril formation
was assessed as thioflavin T fluorescence measured at 444 (excitation)
and 485 (emission) nm. Curve is derived from a nonlinear least squares
fit of triplicate measurements made at intervals as described under
"Experimental Procedures." Data points are omitted for
clarity.
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Fig. 6.
Affinity of N-terminal pro-IAPP fragment for
heparin and heparan sulfate is increased at pH 5.5. Synthetic
peptides corresponding to human pro-IAPP amino acids 1-30 (N-terminal)
or 1-30 with alanine substitutions (K10A/R11A) were applied to a
heparin-agarose (A) or heparan sulfate-Sepharose column
(B), and affinity chromatography was performed as in Fig. 2,
except the Tris-HCl buffer was pH 5.5, as indicated. Arrows
denote elution time of N-terminal pro-IAPP peptide in Tris-HCl at pH
7.5.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical conformation by basic amino acids spaced 13 residues
apart or, in
-strand conformation, 7 residues apart. Although the
basic amino acids close to the N-terminal
Lys10-Arg11 cleavage site (His6,
Arg22, and His29) are not appropriately spaced
according to this model, it is still conceivable that they contribute
to the binding of N-terminal pro-IAPP to heparin/heparan sulfate.
Indeed, the increased affinity for heparin and heparan sulfate of the
N-terminal pro-IAPP peptide at pH 5.5 implies a possible role for the
histidine residues at positions 6 and 29. Also, the disulfide bond
between the cysteines at positions 13 and 18 of pro-IAPP might be
predicted to bring the arginine at position 22 in closer proximity to
the Lys10-Arg11-Lys12 sequence.
However, the uncyclized form of the N-terminal pro-IAPP peptide binds
as well to heparin as the cyclized
form.2 The heparin binding
activity of the N-terminal pro-IAPP peptide therefore does not depend
upon any conformational change induced by formation of the disulfide
bond. Thus, while the
Lys10-Arg11-Lys12 sequence at the
N-terminal cleavage site of pro-IAPP is clearly essential for heparin
binding, whether this sequence is in itself sufficient or whether the
basic residues outside of this sequence are also critical will require
further study using additional mutant peptides.
protein to glycosaminoglycans is known to stimulate
-sheet conformation and aggregation (42), and perlecan has been
shown to stimulate fibril formation from mature human IAPP (25).
-sheet formation, enhancing its tendency to
aggregate. The local accumulation of pro-IAPP bound to perlecan might
form a nidus for amyloid formation to which other amyloidogenic forms of IAPP including the major secreted form, IAPP, could be incorporated following their secretion from neighboring beta cells. In nondiabetic patients (in which islet amyloid is not usually observed) (51), pro-IAPP processing would be expected to be nearly complete (based on
proinsulin (32)), resulting in loss of the pro-IAPP heparin binding
domain prior to exocytosis from the beta cell. Interestingly, the
heparin binding activity of pro-IAPP that we identified was observed
only in the N-terminal and not the C-terminal region of the peptide.
Immunoreactivity for the N-terminal flanking region of pro-IAPP, but
not the C-terminal region, is present in islet amyloid in type 2 diabetic human pancreas (33), raising the possibility that a partially
processed, N-terminally extended pro-IAPP conversion intermediate may
be an important molecule in islet amyloid formation. Whether partially
processed pro-IAPP is secreted in excessive amounts in type 2 diabetes
is unknown; however, beta cells of patients with type 2 diabetes have
disproportionately elevated secretion of both proinsulin and a
partially processed proinsulin conversion intermediate,
des-31,32-proinsulin (32). Moreover, prolonged culture in high glucose
causes marked accumulation of the N-terminally extended pro-IAPP
conversion intermediate in human islets (52) as well as rapid islet
amyloid formation in islets from transgenic mice expressing
amyloidogenic human IAPP (53). If indeed hyperglycemia in type 2 diabetes is associated with excessive secretion of the N-terminal
pro-IAPP conversion intermediate and its subsequent deposition as islet
amyloid, we hypothesize that binding of N-terminal pro-IAPP to basement
membrane heparan sulfate proteoglycans may be an important pathogenic
event in this pathway.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Robert Kisilevsky and John Ancsin for helpful comments in the planning of these experiments.
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada (now Canadian Institutes of Health Research) Grant MT-14682.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Graduate Studentship from the British Columbia
Research Institute for Children's & Women's Health.
§ Supported by a New Investigator award from the Canadian Institutes for Health Research. To whom correspondence should be addressed: British Columbia Research Institute for Children's and Women's Health, 950 West 28th Ave., Vancouver, British Columbia V5Z 4H4, Canada. Tel.: 604-875-2490; Fax: 604-875-3120; E-mail: verchere@interchange.ubc.ca.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M008423200
2 K. Park and C. B. Verchere, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IAPP, islet amyloid polypeptide; PC, prohormone convertase; BSA, bovine serum albumin; Me2SO, dimethyl sulfoxide.
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