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The Journal of Neuroscience, December 1, 2000, 20(23):8745-8749
Insulysin Hydrolyzes Amyloid  Peptides to Products That Are
Neither Neurotoxic Nor Deposit on Amyloid Plaques
Atish
Mukherjee1,
Eun-suk
Song1,
Muthoni
Kihiko-Ehmann2,
Jack P.
Goodman Jr3,
Jan St.
Pyrek3,
Steven
Estus2, and
Louis B.
Hersh1
1 Department of Biochemistry, 2 Department
of Physiology and Sanders-Brown Center on Aging, and 3 Mass
Spectrometry Facility, University of Kentucky, Lexington, Kentucky
40536-0298
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ABSTRACT |
Insulysin (EC. 3.4.22.11) has been implicated in the clearance of
 amyloid peptides through hydrolytic cleavage. To further study the
action of insulysin on A peptides recombinant rat insulysin was
used. Cleavage of both A1-40 and A1-42
by the recombinant enzyme was shown to initially occur at the
His13-His14,
His14-Gln15, and
Phe19-Phe20 bonds. This was
followed by a slower cleavage at the
Lys28-Gly29,
Val18-Phe19, and
Phe20-Ala21 positions. None of
the products appeared to be further metabolized by insulysin. Using a
rat cortical cell system, the action of insulysin on
A1-40 and A1-42 was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown
to prevent the deposition of A1-40 onto a synthetic amyloid. Taken together these results suggest that the use of insulysin
to hydrolyze A peptides represents an alternative gene therapeutic
approach to the treatment of Alzheimer's disease.
Key words:
amyloid peptide metabolism; metallopeptidase; insulysin; A neurotoxicity; A deposition; A cleavage
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INTRODUCTION |
The major pathological feature of
Alzheimer's disease is the presence of senile plaques in the brain of
affected individuals. Although controversy exists whether the formation
of amyloid deposits is the primary cause of Alzheimer's disease, they
contribute to its etiology and progression (Selkoe, 1994 ). These
insoluble amyloid deposits contain as a major constituent amyloid peptides (A peptides) derived by processing of the amyloid precursor
protein (Goldgaber et al., 1987) by and  secretases (Sinha et
al., 1999; Vassar et al., 1999). Considerable effort has been expended in identifying these secretases, the goal being the development of
specific inhibitors that would block the formation of amyloid plaques.
The recent report of an aspartyl protease that appears to be a true secretase (Vassar et al., 1999) provides optimism that this approach
can soon be tested.
Tseng et al. (1999) showed that amyloid formation involves the
deposition of monomeric A. Thus, inhibition of monomeric A aggregation or deposition represents an alternative strategy for the
treatment of Alzheimer's disease. Compounds that prevent A aggregation have been reported (Soto et al., 1996; Tjernberg et al.,
1996; Tomiyama et al., 1996; Wood et al., 1996), and a high throughput
screen has been developed (Esler et al., 1997). Another approach is to
hydrolyze A peptides before they deposit onto amyloid plaques.
Howell et al. (1995) showed that the zinc metallopeptidase neprilysin
(neutral endopeptidase, enkephalinase; EC 3.4.24.11) degraded
A1-40, whereas Iwata et al. (2000) showed
that inhibition of neprilysin in rat brain produces an increase in A1-42 concentration and the formation of
diffuse amyloid plaques. However, it was observed that neprilysin
inhibitors were less effective in altering the
A1-40 concentration, suggesting that
A1-40 might be cleared by a different
mechanism or peptidase (Iwata et al., 2000).
Kurochkin and Goto (1994) showed that another zinc metallopeptidase
insulysin (insulin degrading enzyme, insulinase, EC 3.4.22.11) also
cleaved A1-40, although the products of the
reaction were not identified. In a subsequent study McDermott and
Gibson (1997) confirmed the degradation of
A1-40 by insulysin, identified a number of
putative reaction products, and showed that
A1-40 displayed an IC50
in the low micromolar range. Because this study used partially purified
enzyme, the contribution of contaminating peptidases cannot be ruled
out. Qiu et al. (1998) showed that a secreted form of insulysin was
produced from microglial cells (BV-2) and provided evidence that
primary rat brain cultures and differentiated rat adrenal
pheochromocytoma (PC12) cells expressed a cell surface form of
insulysin (Vekrellis et al., 2000). Recently Perez et al. (2000) showed
that insulysin represents an abundant A degrading activity in human
brain soluble extracts.
In this study we have used homogeneous recombinant rat insulysin to
study the reaction of this peptidase with
A1-40 and A1-42. We
have identified cleavage sites and studied the cleavage reaction and
its effect on the neurotoxic properties of the A peptides and the
ability of A1-40 to deposit onto preformed
synthetic amyloid fibrils. The results of this study suggest that
insulysin may represent an alternative therapeutic approach for the
treatment of Alzheimer's disease.
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MATERIALS AND METHODS |
Materials. A1-40 and
A1-42 were obtained from Bachem (Torrance,
CA). Solutions were prepared by dissolving the peptide in
dimethylsulfoxide (DMSO) to give a stock concentration of 200 µM. The peptide stock was lyophilized and stored at
 80°C until use. The aggregation state of A peptide stock
solutions was checked by electron microscopy (Ray et al., 2000) and
found to be predominantly, if not exclusively, monomeric. For the
in vitro reactions with insulysin, a final concentration of
25 µM A1-40 was
obtained after bringing the lyophilized peptide into solution with
double-distilled water. For cytotoxicity studies A1-40 and A1-42
peptides were dissolved in sterile N2 medium (Life Technologies,
Rockville, MD). Human -endorphin1-31, obtained from the National Institute on Drug Abuse drug supply system,
was dissolved in water to give a stock solution of 300 µM. Trifluoroacetic acid (Sigma, St. Louis, MO)
was diluted into water to produce a 5% working solution.
Expression and purification of recombinant insulysin. A rat
insulysin cDNA, (pECE-IDE), kindly provided by Dr. Richard Roth of
Stanford University (Stanford, CA), was subcloned into the baculovirus-derived vector pFASTBAC (Life Technologies) through BamHI and XhoI restriction sites such that a
His6-affinity tag was attached to the N terminus
of the protein. Generation of recombinant virus and expression of the
recombinant protein in Sf9 cells was performed according to the
manufacturer's directions. For the purification of recombinant
insulysin, a 1:10 (w/v) suspension derived from a 50 ml culture of
viral infected Sf9 cells was prepared in 100 mM
potassium phosphate buffer, pH 7.2, containing 1 mM dithiothreitol
(K-PO4/DTE buffer). The suspension was sonicated 10 times, each burst for 1 sec, using a Branson sonifier (setting 3 at
30%) and then centrifuged at 75,000 × g for 30 min to
pellet cell debris and membranes. The supernatant containing
recombinant rat insulysin was loaded onto a 0.5 ml
nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen, Valencia, CA)
that had been equilibrated with the K-PO4/DTE
buffer. After extensive washing of the column with starting buffer, and
then with 20 mM Imidazole-HCl, pH 7.2, the enzyme
was eluted with 0.1 M Imidazole-HCl, pH 7.2. The
enzyme was further purified over a 1 ml Mono-Q anion exchange column (Pharmacia Biotech, Piscataway, NJ) in 20 mM
phosphate buffer, pH 7.2. A linear salt gradient of 0-0.6
M KCl, equivalent to 60 column volumes, was
applied to the column with the enzyme eluted at 0.28 M KCl. SDS-PAGE of the insulysin was conducted on
a 7.5% gel.
Insulysin activity determination. Insulysin activity was
assayed by measuring the disappearance of -endorphin by isocratic reverse-phase HPLC (Safavi et al., 1996). A 100 µl reaction mixture containing 40 mM potassium phosphate buffer, pH 7.2, 30 µM -endorphin, and enzyme was incubated for 15 min at
37°C. The reaction was stopped by the addition of 10 µl of 5%
trifluoroacetic acid to give a final concentration of 0.5%. The
reaction mix was loaded onto a C4 reverse-phase
HPLC column (Vydac, Hisperia, CA), and products were resolved
isocratically at 32% acetonitrile. The -endorphin peak was detected
by absorbance at 214 nm using a Waters 484 detector. The reaction was
quantitated by measuring the decrease in the -endorphin peak area.
Determination of sites of cleavage of A peptides.
Purified insulysin was incubated with 25 µM
A1-40 in 40 mM potassium phosphate buffer, pH 7.2, at 37°C for 1 hr. The reaction products were loaded onto a C4 reverse-phase HPLC column
and products resolved using a linear gradient of 5-75% acetonitrile
over 65 min. Products were detected by absorbance at 214 nm using a
Waters 484 detector, and individual product peaks were collected
manually. Product analysis was also conducted on an intact reaction
mixture in which products were not resolved by HPLC. Products were
identified by matrix-assisted laser desorption ionization time of
flight mass spectrometry (MALDI-TOF-MS). The reaction of insulysin with
A1-42 was conducted in a similar manner with
products identified by MALDI-TOF-MS directly from reaction mixtures.
AB1-40 deposition assay. amyloid deposition
assays were conducted as described by Esler et al. (1999). Briefly, 96 well microtiter plates precoated with aggregated amyloid
1-40 (QCB/Biosource, Hopkinton, MA) were
additionally coated with 200 µl of a 0.1% bovine serum albumin
solution in 50 mM Tris-HCl, pH 7.5, for 20 min to prevent
nonspecific binding. For measuring A1-40
deposition in the presence or absence of insulysin, a 150 µl solution
of 0.1 nM 125I labeled
A1-40 in 50 mM Tris-HCl, pH 7.5, was added to the precoated well and incubated for 4 hr. When added,
insulysin (0.5-500 ng) was placed directly in the well at zero time.
The reaction was stopped by washing off excess undeposited radiolabeled A1-40 with 50 mM Tris-HCl, pH
7.5. The radiolabel deposited onto the washed well was counted in a
gamma counter. In a variation of this protocol, insulysin was
preincubated with 1 nM
125I-A1-40 for
60 min and then added to the deposition assay.
Neuoroprotection assays. Neurotoxicity assays were performed
as described by Estus et al. (1997) using embryonic day 18 rat fetuses
to establish primary rat cortical neuron cultures. Rat brain cortical
cells were initially cultured in AM0 media for 3-5 hr in 16 well chamber slides (Nalge Nunc International, Rochester, NY) precoated with polyethyleneimine at a density of ~1 × 105 cells per well. The culture was
enriched in neurons by replacement of the AM0
media with DMEM (Life Technologies) containing 100 U/ml
penicillin, 100 µg/ml streptomycin, and 2% B27 serum supplement (Life Technologies).
Cells were treated with A peptides and then fixed with 4%
paraformaldehyde for 15 min at room temperature. After washing the
cells with PBS, they were then stained with Hoechst 33258 at 1 µg/ml
for 10 min. Neurons were then visualized by fluorescence microscopy.
Those cells with uniformly dispersed chromatin were scored as
survivors, whereas those cells containing condensed chromatin were
scored as nonsurvivors. Readings were typically taken in triplicate
with a minimum of 250 neurons scored from each well. Cells treated as
described above were visualized using a Nikon microscope equipped with
a Hoffman modulation contrast lens. Statistical analysis was performed
on the samples using ANOVA.
Immunofluorescence. The presence of aggregated
A1-40 was detected in the neuronal cultures
using the monoclonal antibody 10D5 (Walker et al., 1994) at a 1:100
dilution in 5% goat serum in PBS. After an overnight incubation at
4°C with this primary antibody, the wells were rinsed with PBS and
incubated with a goat anti-mouse secondary antibody conjugated to Cy3
(Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:250 in 5%
goat serum in PBS. The wells were incubated at room temperature for 60 min and then after further washing with PBS, cells were examined under
a fluorescence microscope.
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RESULTS |
To characterize the reaction of insulysin with the A peptides,
recombinant rat enzyme containing an N-terminal
His6 affinity tag was expressed in
baculovirus-infected Sf9 cells. Expression of the enzyme in this system
was high, as evidenced by the ability to see insulysin protein in a
crude extract by SDS-PAGE (Fig. 1).
Purification of the recombinant enzyme was achieved by chromatography on a Ni-NTA-agarose column producing highly purified enzyme followed by
chromatography on a Mono-Q column, which produced homogeneous enzyme
(Fig. 1). The specific activity of the recombinant enzyme (2.6 µmol · min 1 · mg1)
was comparable to enzyme purified from a thymoma cell line, EL-4 (3.3 µmol · min1 · mg1),
and thus the presence of the His6 affinity tag
had no discernable effect on enzyme activity.
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Figure 1.
Purification of recombinant rat insulysin.
Insulysin was purified as described in Materials and Methods, and 15 µg aliquots from various stages of purification were analyzed by
SDS-PAGE on a 7.5% gel stained with Coomassie Blue. A,
Sf9 cell extract. B, Nonbound proteins from the
Ni-NTA-agarose column. C, Protein eluted from the
Ni-NTA-agarose column with 20 mM imidazole.
D, Protein eluted from the Ni-NTA-agarose column with
100 mM imidazole. E, Protein eluted from the
Mono-Q column. The position of molecular weight markers (myosin, 200 kDa; -galactosidase, 116 kDa; phosphorylase B, 97.4 kDa; bovine
serum albumin, 66 kDa; and ovalbumin, 45 kDa) is shown on the
left.
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To delineate the sites of cleavage of the
A1-40 peptide by insulysin, the peptide was
incubated with varying concentrations of the enzyme for 1 hr at 37°C,
and then products were resolved by gradient reverse-phase HPLC. With 50 ng of insulysin, the lowest enzyme concentration used, three major
cleavage sites at
His14-Gln15
(peak 1),
His13-His14
(peak 2), and
Phe19-Phe20
(peak 4 and peak 7) were discernible (Table
1, Fig. 2).
In addition, minor cleavage sites at
Lys28-Gly29
(peak 5) and
Phe20-Ala21
(peak 6) were observed. When the amount of insulysin was increased to
250 ng, each of the products seen with 50 ng of enzyme increased, and
an additional product corresponding to cleavage at
Val18-Phe19
(peak 3) was observed. Further increasing insulysin to 500 ng showed a
continued increase in each of the products. The same products were seen
when A1-40 was treated with 500 ng of insulysin and analyzed by MALDI-TOF-MS without separation of the reaction products. It is interesting to note that one product peak
A14-40 was not observed, whereas other
product peaks were not apparent until after substantial metabolism had
occurred. For example, A1-14 can be seen in
the digest using 50 ng of insulysin, whereas the product corresponding
to the C-terminal half of this cleavage,
A15-40, is not seen in the 50 ng reaction,
but is observed with the 250 ng of enzyme. This is in part attributed
to the hydrophobic nature of the C-terminal peptides and their greater
retention times, which produces HPLC peak broadening and decreased
sensitivity. The overall cleavage profile is illustrated in Figure
3.
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Figure 2.
HPLC profile of products generated from the
cleavage of A1-40 by insulysin. Varying amounts of
recombinant rat insulysin were incubated with 25 µM
A1-40 for 30 min at 37°C. Cleavage products were
separated by a 5-75% gradient of acetonitrile on a C4
reverse-phase HPLC column. Product peaks are numbered according to
their order of elution. The peaks designated Ca and
Cb refer to contaminants in the A1-40
solution. These are not reacted on by insulysin, as is seen by their
invariant peak areas in all the traces. A,
A1-40 alone. B, A1-40
incubated with 50 ng of insulysin. C,
A1-40 incubated with 250 ng of insulysin.
D, A1-40 incubated with 500 ng of
insulysin. The HPLC scans are skewed ~2 min to the left to permit
overlapping peaks to be viewed. The time scale refers to trace
A.
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Figure 3.
Positions of cleavage within the
A1-40 and A1-42 sequences. Primary
cleavage sites are noted with the thick arrows.
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The A1-42 peptide was incubated with
insulysin in an identical manner as with
A1-40, and the products were analyzed by
MALDI-TOF mass spectrometry without prior separation by HPLC. Product
peaks corresponding to cleavage at the
His13-His14,
His14-Gln15,
Phe19-Phe20,
and
Phe20-Ala21
positions were observed. These results indicate that both
A1-40 and A1-42 are
cleaved at the same sites. The rate of cleavage of 25 µM
A1-40 was measured as 1.2 µmol · min1 · mg1
enzyme, which indicates that the A peptides are good substrates for insulysin.
The products of the action of insulysin on the A peptides produces
relatively large fragments. Because the peptide
A25-35, which is derived from
A1-40, is neurotoxic, it is possible that the
products of insulysin action on the A peptides could be toxic to
neurons. To test this, rat cortical neurons were treated with A
peptides in the presence and absence of insulysin. Preliminary experiments were performed to obtain a suitable A peptide
concentration that would show a significant cytotoxic effect, as there
are batch to batch variations in the ability of the A peptides to
mediate cytotoxic effects on cells in culture. These experiments
established 30 µM A1-40 and 25 µM A1-42 as reasonable peptide concentrations that produce ~70 and 80% loss of cortical neurons, respectively, in 48 hr.
The cell-based assay using primary rat cortical neurons was used to
determine whether the insulysin cleavage products of the A peptides
were themselves neurotoxic. Recombinant insulysin at concentrations
ranging from 0.5 to 5000 ng was added simultaneously with the A
peptides to the cortical cultures. When added directly to the cultures,
as little as 50 ng of insulysin was effective in sparing the neurotoxic
effects of A1-40 (Fig.
4A), whereas 500 ng of
insulysin was effective in sparing the neurotoxic effects of
A1-42 (Fig. 4B). This
effect of insulysin is illustrated in Figure
5 in which cells were either stained with
Hoechst 33258 to visualize DNA (A-D), with the A
antibody 10D5 to visualize cell-associated A
(E-H), or visualized directly by Hoffman modulation microscopy (I-L). Using this phase-contrast
microscopy, it can be seen that A1-40 caused
the cells to appear shrunken (K) as compared to
control cells, which appear rounded (I).
A1-40 induced chromatin condensation, which
appears as small rounded nuclei (C), and A
cellular accumulation, which appears as a bright layering over the
cells (G), is not evident in untreated cells (A, E). Cells to which insulysin was added along
with A1-40 more closely resembled untreated
cells (D, H, L). Also shown in Figure 5 are
controls in which cells were treated with insulysin alone
(B, F, J).
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Figure 4.
Effect of insulysin on the neurotoxic effects of
A peptides. Purified insulysin was added with A1-40
(30 µM) or A1-42 (25 µM) to
primary cortical neurons, and incubation continued for an additional 48 hr. The neurotoxic effect of the A peptides was determined as
described in Materials and Methods. The insulysin and heat-inactivated
insulysin controls used 5000 ng of enzyme. A, Effect of
incubation with insulysin on the neurotoxic effects of
A1-40. B, Effect of incubation with
insulysin on the neurotoxic effects of A1-42.
*p < 0.01 relative to the A-treated sample as
determined by ANOVA.
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Figure 5.
Insulysin protects against A1-40
mediated neurotoxicity. Rat cortical neurons were treated as described
in Figure 4 in the presence or absence of 50 ng of insulysin. Cells
were stained with Hoechst 33258 (A-D) or with
the A antibody 10D5 (E-H). Hoffman modulation
contrast micrographs are shown in I-L.
A, E, and I show untreated
neurons. B, F, and J show
neurons with 50 ng of insulysin added. C,
G, and K show neurons treated with 30 µM A1-40. D,
H, and L show neurons treated with 50 ng
of insulysin and 30 µM A1-40.
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During the progression of Alzheimer's disease, monomeric A peptides
are deposited onto senile plaques. To test whether insulysin is able to
prevent the deposition of the A1-40 peptide, a model system was used in which the deposition of radiolabeled A1-40 onto a synthetic amyloid plaque
(synthaloid) is followed (Esler et al., 1999). As seen in Figure
6A, addition of
insulysin at 0.5-500 ng with radiolabeled
[125I]A1-40
shows that 50 ng of insulysin is able to prevent the deposition of
radiolabeled A1-40. Figure
6B shows that preincubation of insulysin with
radiolabeled
[125I]A1-40
for 60 min before adding it to the wells also shows that 50 ng of
insulysin is able to prevent the deposition of radiolabeled A1-40 onto the synthetic amyloid. We also
conducted an experiment in which
[125I]A1-40
was first deposited onto the synthetic amyloid and then treated with
insulysin to see if the enzyme could degrade preaggregated
A1-40. After a 24 hr incubation with 5 µg of insulysin, no radioactivity was released, indicating that insulysin does not degrade aggregated A peptides.
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Figure 6.
Insulysin inhibits the deposition of
A1-40 onto synthetic amyloid plaques. A,
Effect of incubation with insulysin on the deposition of
A1-40. A1-40 (0.1 nM) was
mixed with the indicated amount of purified insulysin and then added to
synthaloid in 96 well plates. Deposition was permitted to occur over a
4 hr time period. B, Effect of preincubation with
insulysin on the deposition of A1-40.
A1-40 (1 nM) was preincubated for 60 min
with the indicated amount of purified insulysin. The incubation
mixtures were then added to synthaloid in 96 well plates, and
deposition was permitted to occur over a 4 hr time period.
*p < 0.01 as determined by ANOVA.
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DISCUSSION |
The balance between the anabolic and catabolic pathways in the
metabolism of the A peptides is a delicate one. Although
considerable effort has focused on the generation of the A peptides,
until recently considerably less emphasis has been placed on the
clearance of these peptides. Removal of extracellular A peptide
appears to proceed through two general mechanisms: cellular
internalization and extracellular degradation by neuropeptidases.
Apparently neither of these mechanisms is adequate in Alzheimer's
disease. Interest in the mechanism of cellular internalization stems
from the apparent involvement of apolipoprotein E and
 -2-macroglobulin in this process (Narita et al., 1997; Kang et al.,
1997; Hughes et al., 1998; Blacker et al., 1998).
A number of neuropeptidases have been suggested to be involved in the
extracellular degradation of A peptides, and these include
neprilysin (Howell et al., 1995), insulysin (Kurochkin and Goto, 1994;
McDermott and Gibson, 1997; Qiu et al., 1998), and the plasmin system
(Tucker et al., 2000). Studies by Iwata et al. (2000) showed that
inhibition of neprilysin in rat brain led to increased levels of
A1-42 and the formation of diffuse plaques.
Interestingly, A1-40 levels did not increase
as a consequence of neprilysin inhibition, suggesting that a different peptidase may be responsible for A1-40
metabolism. Previous studies have shown that the zinc metalloprotease
insulysin (insulin degrading enzyme) is able to cleave
A1-40 (Kurochkin and Goto, 1994; McDermott
and Gibson, 1997; Qiu et al., 1998), making this a candidate enzyme for
its clearance. Perez et al. (2000) showed that the insulysin activity
was decreased in the soluble fraction derived from human Alzheimer
brains compared to aged matched controls. They suggested this decrease
could contribute to increased A accumulation in Alzheimer's disease.
The use of neuropeptidases such as neprilysin or insulysin to remove
extracellular A peptides represents a potential treatment for
Alzheimer's disease. However, for a peptidase to be useful, it must be
established that its action eliminates the amyloidogenic properties of
A peptides. In this study we have shown that insulysin cleaves both
A1-40 and A1-42 at
the
His13-His14,
His14-Gln15,
and
Phe19-Phe20
bonds as initial cleavage sites. Although the exact substrate specificity of insulysin is still unclear, it has been observed that
insulysin can cleave at the C terminus of basic and hydrophobic amino
acid residues. Thus, the cleavage pattern obtained with A1-40 and A1-42 is
consistent with this specificity. Other cleavage sites that appear
using higher concentrations of insulysin are at the
Lys28-Gly29,
Val18-Phe19,
and
Phe20-Ala21
positions. These cleavage sites are also consistent with the known
substrate specificity of the enzyme.
The cleavage products observed with insulysin indicate distinct
cleavage events and not products derived from secondary cleavage of an
initial product. That is, no fragment was observed lacking an intact N
terminus, the C-terminal fragment corresponding to each N-terminal
fragment was seen in all but one case, and products increased with an
increasing concentration of insulysin.
Neuronal cell cultures are susceptible to the toxic effects mediated by
A1-40 and A1-42. We
have used this neuronal cell culture system to establish that the
products of the insulysin-dependent cleavage of
A1-40 and A1-42
produces products that are not in themselves neurotoxic. This is an
important point if one were to consider the use of insulysin in the
treatment of Alzheimer's disease.
Related to cellular toxicity, A peptides are able to deposit onto an
existing matrix of peptides in what is thought to lead to an increase
in the size of senile plaques and consequently to the progression of
Alzheimer's disease. In a model system, Esler et al. (1997) have shown
that the deposition of A1-40 onto a preformed
synthaloid matrix mimics the in vivo deposition of A
peptides onto the brain cortex. Using this model, we have shown that
insulysin cleavage of A1-40 prevents the
deposition of the A peptides onto the synthaloid. This suggests that
insulysin may be able to prevent the formation and growth of senile
plaques in Alzheimer's disease patients.
In summary we have established that the insulysin-dependent cleavage of
the A peptides leads to the loss of both their neurotoxic properties
as well as their ability to contribute to plaque formation and growth.
The use of insulysin and other peptidases to degrade extracellular A
peptides represents a new approach toward the treatment of Alzheimer's disease.
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FOOTNOTES |
Received June 28, 2000; revised Sept. 8, 2000; accepted Sept. 15, 2000.
This research was supported in part by National Institute on Drug Abuse
Grants DA 02243 and DA 07062 and National Institute on Aging Grant AG
05893. We thank Dr. Richard Roth of Stanford University for providing
us with the cDNA clone to rat insulysin and for insulysin antisera, Dr.
John Maggio and Jeffrey R. Marshall of the University of Cincinnati for
helping us establish the A deposition assay, and Drs. Mark Lovell,
Chengsong Xie, and William Markesbery of the Sanders-Brown Center on
Aging, University of Kentucky for helping us in preliminary neuronal
toxicity studies.
Correspondence should be addressed to Dr. Louis B. Hersh, Department of
Biochemistry, University of Kentucky, Chandler Medical Center, 800 Rose
Street, Lexington, KY 40536-0298. E-mail: lhersh{at}pop.uky.edu.
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TOP
ABSTRACT
INTRODUCTION
