Carbohydrate Polymers xx (2005) 1–5
Chitooligosaccharides as a novel b-secretase inhibitor
Hee-Guk Byuna, Yong-Tae Kimb, Pyo-Jam Parkc, Xinli Linb, Se-Kwon Kima,*
Department of Chemistry, Pukyong National University, Busan 608-737, South Korea
Functional Proteomics Laboratory, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
Department of Biotechnology, Konkuk University, Chungju 380-701, South Korea
Received 24 September 2004; revised 27 April 2005; accepted 3 May 2005
Nine kinds of hetero-chitooligosaccharides (hetero-COSs) with different degrees of deacetylation and molecular weights were prepared
using an ultrafiltration (UF) membrane reactor system. In addition, their sulfated derivatives were also synthesized by a method using
trimethylamine-sulfur trioxide to investigate the functional group of COSs on b-secretase inhibitory activity. 90-MMWCOSs-I, which are
90% deacetylated COSs passed through the 5 kDa membrane but not passed through the 3 kDa membrane, exhibited the highest b-secretase
inhibitory activity (25–42 mM) based on molecular weight of 3 and 5 kDa. The inhibition pattern of the inhibitor was found to be a noncompetitive by Dixon plot, and Ki of 90-MMWCOSs-I was 3.87–6.47 mM. Therefore, the data of this research suggest that 90-MMWCOSs-I
is a good candidate target molecule to inhibit b-secretase.
q 2005 Published by Elsevier Ltd.
Keywords: Alzheimer’s disease; b-Secretase inhibitor; Chitooligosaccharide (COS)
1. Introduction
Alzheimer’s disease (AD) is thought to be caused by the
progressive brain accumulation of b-amyloid (Ab) peptides
into fibrillar aggregates and insoluble plaques resulting
severe memory loss and neuronal cell death (Selkoe, 2001).
The Ab peptides are generated by endoproteolysis of the
b-amyloid precursor protein (b-APP) by two proteolytic
enzymes, b- and g-secretase. The b-secretase generates
the N-terminus of Ab peptides by cleaving APP at Met670/
Asp671, while g-secretase cleaves the C-terminus of the
peptides by proteolysis either at Val711 or Ala713, the
resultant Ab peptides is either 40 or 42 amino acid residues
in length (Dorrel, 2000). The Ab42 peptide is the most
abundant and which play critical roles on the induction of
AD (Zohar, Cavallaro, Agata, & Alkon, 2003).
b-Secretase is an aspartic protease and also known as
BACE (the b-site APP-cleaving enzyme). This enzyme
cleaves an easily accessible site at the luminal side of
* Corresponding author. Tel.: C82 51 620 6375; fax: C82 51 628 8147.
E-mail addresses: [email protected] (S.-K. Kim), [email protected] (S.-K. Kim).
0144-8617/$ - see front matter q 2005 Published by Elsevier Ltd.
b-APP, and its activity is the rate-limiting step in Ab peptide
production in vivo (Vassar et al., 1999). b-Secretase activity
is present in the majority of cells and tissues of the body
(Haass et al., 1992). The maximal activity is found in neural
tissues and cell lines (Seubert et al., 1993; Zhao et al., 1996).
b-Secretase is widely expressed in various tissues and cell
lines, but would be at higher levels in neurons of the brain.
b-Secretase is a major target for screening of inhibitors
since it occupies the initial step in the pathological cascade
of AD. Thus, the inhibition of b-secretase acting in vivo
may reduce the production of Ab peptides expecting that it
slow or halt the progression of AD.
Recently, two transition-state analog inhibitors of
b-secretase were reported on the basis of the model on the
cleavage site on the b-secretase of the Swedish mutation
(Zhao et al., 1996). The first inhibitor, P10-P4’StatVal,
contains Asn at P2, statine group at P1, and Val at P1’, and
has an IC50 value of w30 nM (Sinha et al., 1999). The other
type or reported inhibitors are OM99-1 and OM99-2.
OM99-2 has an IC50 of w1.6 nM and is P4-P4 0 with
AsnLeu at P2-P1, Ala at P1 0 , and a hydroxyethylene isostere
between P1 and P1 0 (Ghosh et al., 2001). However, the
therapeutic potential of above inhibitors might be restricted
by their higher molecular weight (MW, about 1100 Da) and
numerous peptide bonds (Ghosh et al., 2001). In order to be
H.-G. Byun et al. / Carbohydrate Polymers xx (2005) 1–5
a good candidate for therapeutic potential, the molecular
weights of inhibitors are preferably smaller than 700 Da, so
large peptide-base inhibitors are not viable drug candidates.
Thus, the peptidic inhibitors and the metabolites of plants
and microbes which have relatively low molecular weights
and lipophilicity might be good b-secretase inhibitors as
drug candidates (Dorrel, 2000).
In previous studies, potent peptidic inhibitors of the
b-secretase have already been identified (Ghosh et al., 2001;
Vassar et al., 1999; Zohar et al., 2003). However, there was
not published report of carbohydrates as b-secretase
inhibitors. Chitosan is a deacetylated polymer of N-acetyl
glucosamine, which is obtained after alkaline deacetylation
of the chitin derived from the exoskeletons of crustaceans
and arthropods. It has shown to possess a hypocholesterolemic effect (Jennings, Boleyn, Bridges, Wood, &
Anderson, 1988; LeHoux & Grondin, 1993; Maezaki
et al., 1993), an immunomodulating function (Lim et al.,
1997), and a hypoglycemic effect (Miura, Usami, Tsuura,
Ishida, & Seino, 1995). However, recent studies on chitosan
have attracted interest for converting chitosan to its
oligosaccharides, because the oligosaccharides are not
only water soluble but also reported to have special
functional properties such as antitumor activity (Jeon &
Kim, 2002; Suzuki et al., 1986), immunostimulating effect
(Jeon & Kim, 2001), antimicrobial activity (Hadwiger &
Beckman, 1980; Kendra, Christian, & Hadwiger, 1989;
Park, Je, Byun, Moon, & Kim, 2004), radical scavenging
activity (Je, Park, & Kim, 2004).
In the present study, b-secretase inhibitory activity of
hetero-chitooligosaccharides (hetero-COSs) prepared from
partially different deacetylated chitosans was investigated,
and the inhibition pattern was also determined using COSs
with molecular weights between 3000 and 5000 Da
prepared from 90% deacetylated chitosan, which exhibited
the highest b-secretase inhibitory activity as an inhibitor
determined by using Dixon plots.
2. Materials and methods
2.1. Materials
Chitin prepared from crab shells was donated by Kitto
Life Co. (Seoul, Korea). The chitosanase (35,000 U/g
protein) derived from Bacillus sp. was purchased from
Amicosen Co. (Jinju, Korea), and cellulose was donated by
Pacific Chemical Co. (Seoul, Korea). An ultrafiltration (UF)
membrane reactor system (Minitane) for production of
hetero-COSs was from Millipore Co. (Bedford, MA, USA).
Fluorogenic substrate FS-1, NH2-Arg-Glu (EDANS)Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DABCYL)-ArgCOOH (MW, 2005.0 kDa), was synthesized at SynPep
(Dublin, CA) and FS-2 (MCA) Ser-Glu-Val-Asn-Leu-AspAla-Glu-Phe-Lys(DNP) (MW, 1533.6 kDa) was synthesized at the Molecular Biology Resource Center,
University of Oklahoma Health Sciences Center, using an
Applied Biosystems Peptide Synthesizer 430A (Foster,
CA). In addition, b-secretase (198 U/mg-protein) was
purchased from the Molecular Biology Resource Center,
University of Oklahoma Health Sciences Center. All other
reagents were the highest grade commercially available.
2.2. Preparation of hetero-COSs and synthesis
of sulfated hetero-COSs
Three kinds of partially deacetylated chitosans, 90, 75
and 50% deacetylated chitosan, were prepared from crab
chitin by N-deactylation with 40% (w/v) sodium hydroxide
solution for different durations based on the method of Park,
Je, and Kim (2004), and hetero-COSs, which are COSs
prepared from 90, 75, and 50% deacetylated chitosans, were
prepared by hydrolysis of hetero-chitosans in an UF
membrane reactor system according to the method of
Park, Lee, and Kim (2004). The sulfated COSs were
synthesized according to the method of Park, Je, Jung, Ahn,
and Kim (2004), and dialyzed exhaustively against distilled
water using an electronic dialyzer (Micro Acilyzer G3,
Asahi Chemical Industry Co.(Tokyo, Japan). The dialyzer
membrane used was Aciplex Cartridge (AC-230-400).
2.3. Assay for b-secretase inhibitory activity
The inhibition assay of b-secretase was performed by the
method of Ermolieff et al. (2000). Assay for b-secretase
inhibitory activity was performed in 0.1 M sodium acetate
buffer (pH 4.0) at 37 8C with 10% dimethyl sulfoxide
(DMSO) using substrate 300 mM FS-1. The reaction was
initiated with 1780 ml buffer, 180 ml DMSO and 20 ml
enzyme in cubic cell for 10 min. And then 20 ml substrate
was added to it placed in Fluorescence spectrophotometer
(Perkin–Elimer LS50B, Beaconsfield Bucks, UK). For
kinetic assays, initial velocity and steady state were
strictly maintained. Substrate concentrations in the range
0.6–6.0 mM were used. The increase of fluorescence
intensity produced during substrate hydrolysis was studied
in a continuous assay using Fluorescence spectrophotometer. All data were measured as mean of triplicate. An
excitation wavelength of 350 nm and an emission
wavelength of 490 nm were used to monitor the hydrolysis
of substrate FS-1. The IC50 value was defined as a
concentration of the b-secretase inhibitor that is required
to inhibit 50% of the inhibitory activity. In addition,
inhibition constants (Ki) of b-secretase inhibitors were
calculated by Dixon plots.
3. Results and discussion
Recently, chitosan and its oligomers have reported to
possess various bioactive activities, and their properties are
presumed to be dependent on their degree of deacetylation
H.-G. Byun et al. / Carbohydrate Polymers xx (2005) 1–5
Fig. 1. b-Secretase inhibitory activity of the chitooligosaccharides
fractionated by the molecular weight distribution and the degree of
deacetylation. 50, 75, and 90% deacetylated chitooligosaccharides is shown
50-COS, 75-COS, 90-COS, respectively. Molecular weight distributions of
each COSs is shown LMWCOSs, below 1 kDa; MMWCOSs, 1–5 kDa;
HMWCOSs, 5–10 kDa.
HMWCOSs : 5~10 kDa
MMWCOSs : 1~5 kDa
and 90-COS sulfates in all of the molecular weight
distributions are shown in Fig. 2. The 90-COS sulfates
showed a lower inhibitory activity compared with that of
90-COSs. Above results indicated that the deacetylation
and sulfation at C-2 position of COSs have an effect on
b-secretase inhibitory activity. Further amine group at C-2
position was shown to be beneficial for the b-secretase
inhibitory activity. Also the 90-MMWCOSs were fractionated into 90-MMWCOSs-I, that are 90-COSs passed
through the 5 kDa membrane but not passed through the
3 kDa membrane, and 90-MMWCOSs-II, that are 90-COSs
passed through the 3 kDa membrane but not passed
through the 1 kDa membrane by UF membrane reactor
system with MWCO 1 and 3 kDa membranes. b-Secretase
inhibitory activity of N-acetylglucosamine, D-glucosamine,
90-MMWCOSs-I, and 90-MMWCOSs-II are shown in
Fig. 3. N-acetylglucosamine and D-glucosamine were
LMWCOSs : ~1 kDa
Fig. 2. Comparison of b-secretase inhibitory activity of chitooligosaccharides and chitooligosaccharide sulfates. Molecular weight distributions
of each 90% deacetylated COSs is shown 90-LMWCOSs, below 1 kDa;
90-MMWCOSs, 1–5 kDa; 90-HMWCOSs, 5–10 kDa.
COS sulfates
β-secretase inhibitory activity (%)
β-secretase inhibitory activity (%)
and molecular weights. However, there is no information on
bioactive properties for both degree of deacetylation and
their molecular weights. Therefore, nine different kinds of
hetero-COSs were prepared by an UF membrane reactor
system to investigate a biological activity based on their
degree of deacetylation and molecular weights. The
deacetylated chitosans of 90, 75, and 50% were hydrolyzed
and fractionated by passing them through three UF
membranes of molecular weight cut-off (MWCO) 10, 5,
and 1 kDa, respectively. The hetero-COSs were named
90-HMWCOSs, 75-HMWCOSs, and 50-HMWCOSs, that
are 90, 75, and 50% deacetylated COSs passed through the
MWCO 10 kDa membrane but not passed through the
5 kDa membrane, 90-MMWCOSs, 75-MMWCOSs, and
50-MMWCOSs, that are 90, 75, and 50% deacetylated
COSs passed through the 5 kDa membrane but not passed
through the 1 kDa membrane, and 90-LMWCOSws,
75-LMWCOSs, and 50-LMWCOSs, that are 90, 75,
and 50% deacetylated COSs passed through the 1 kDa
membrane, respectively.
The b-secretase inhibitory activity of nine different kinds
of hetero-COSs was shown in Fig. 1. Among different
deacetylated COSs, 90% deacetylated COSs showed
approximately three times higher b-secretase inhibitory
activity than those of 75 and 50% deacetylated COSs in
all of the molecular weight distributions. In addition,
90-MMWCOSs exhibited the highest inhibitory activity
compared with that of the other hetero-COSs. These results
indicated that b-secretase inhibitory activity of hetero-COSs
was different on degree of deacetylation compared with
molecular weight distributions of COSs.
In addition, 90-COS su lfates were synthesized from
90-COSs that exhibited the highest inhibitory activity,
to investigate the effect of functional group of COSs on
b-secretase inhibitory activity. In our previous study, sulfate
groups were identified at the positions of C-2, C-3 and C-6
(Park et al., 2004). The inhibitory activities of 90-COSs
Fig. 3. b-Secretase inhibitory activity of N-acetylgulcosamine, glucosamine
and 90% deacetylated chitooligosaccharides fractionated by molecular
weight distributions. LMWCOSs, below 1 kDa; MMWCOSs-I, 3–5 kDa;
MMWCOSs-II, 1–3 kDa; HMWCOSs, 5–10 kDa.
H.-G. Byun et al. / Carbohydrate Polymers xx (2005) 1–5
strongly suggests that 90-MMWCOSs-I might be bind to
either another regulatory site or subsite of b-secretase. As
summarized in Table 1, the inhibition constant (Ki) of the
90-MMWCOSs-I was about 3.87–6.47 mM calculated by
Dixon plot.
In conclusion, b-secretase inhibitory activity of COSs
was dependent on relatively high degree of deacetylation,
and the 90-MMWCOSs-I showed the higher b-secretase
inhibitory activity. Therefore, 90-MMWCOSs-I is a good
candidate target molecules as a b-secretase inhibitor.
[S] = 0.6 µM
1/V (F.U./sec)–1
[S] = 1.5 µM
[S] = 3.0 µM
[S] = 6.0 µM
Inhibitor concentration (µg/ml)
Fig. 4. Dixon plot for determining inhibitor constants of 90-MMWCOSs-II
against b-secretase with substrate concentration.
exhibited lower inhibitory activities as much as 55 and 45%
compared with that of 90-MMWCOSs-I, respectively.
However, the inhibitory activity of D-glucosamine was
higher than that of N-acetylglucosamine with N-acetyl
group at C-2 position. The b-secretase inhibitory activity of
90-MMWCOSs-I was approximately 20% higher than that
of 90-MMWCOSs-II. We further characterized the inhibitory concentration of 90-MMWCOSs-I, and the IC50 value
was determined to be 25–42 mM based on molecular
weights of 3 and 5 kDa. In recent studies, peptidic inhibitors
are targeted as b-secretase inhibitors. Shuto et al. (2003)
elucidated that a synthesized octapeptide (Glu-Val-LeuPns-Asp-Ala-Glu-Phe) showed the highest activity (IC50
value Z0.41 mM) among the tested peptidic inhibitors. In
spite of the highest inhibition efficiency, they reported that
the octapeptide is needed to reduce the size of molecular
weight to overcome the metabolic instability. Kimura et al.
(2004) recently reported the synthesis of a small-sized and
highly potent b-secretase inhibitor KMI-370 (IC50 value Z
3.4 nM) using octapeptide as a lead compound.
Non-peptidic inhibitors extracted from green tea exhibited the IC50 values of 1.6–4.5 mM (Jeon, Bae, Seong, &
Song, 2003). The inhibitory activity of this present study of
the 90-MMWCOSs-I was less than that of peptidic
inhibitors. However, this is the first report on the target
inhibitors using COSs as non-peptidic inhibitors.
b-Secretase inhibition pattern against 90-MMWCOSs-I
was found to be non-competitive at the active site of
b-secretase determined using Dixon plot (Fig. 4). Thus, it
Table 1
Inhibitor constants (Ki) of 90-MMWCOSs-II (MW 3–5 kDa) calculated by
Dixon plot
Linear formula
of plot
Ki (M)
This research was supported by a grant (P-2004-01) from
Marine Bioprocess Research Center of the Marine Bio 21
Center funded by the Ministry of Maritime Affairs &
Fisheries, Republic of Korea
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