We have found a-anomers that possess potent antitumor activ-ity but do not have many of the serious side-effects of Keywords mammalian DNA polymerases; MT depolymerization activity of MC
Trang 1A new molecular target of sulfoquinovosylacylglycerols novel
antitumor and immunosuppressive agents
Satoko Aoki1,2, Keisuke Ohta1, Takayuki Yamazaki1, Fumio Sugawara1,2and Kengo Sakaguchi1,2
1 Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan
2 Genome and Drug Research Center, Tokyo University of Science, Noda, Chiba, Japan
Several kinds of synthetic sulfoquinovosylacylglycerols
(SQAGs) may be potent and clinically promising
agents for cancer chemotherapy and
immunosuppres-sion [1,2] However, the molecular targets of SQAGs
are ambiguous The aim of the present study was to
identify molecular targets that could be of significance
Earlier, effects of SQAGs were found and reported
independently by two laboratories screening directly
for antitumor agents in vivo [1] and for mammalian
DNA polymerase inhibitors in vitro [3–5] One
mole-cular target of SQAGs is thus DNA polymerases [6]
but there is a strong evidence for other targets [7–9]
SQAGs are found as natural compounds in higher
plants [3], sea algae [4,5] and sea urchins [1] They
have been reported to have a wide range of bioactivi-ties: antiviral activity against human immunodeficiency virus (HIV-1) [7], P-selectin receptor inhibition [8], antitumor activity [1], tumor cell growth inhibition [6] and immunosuppressive activity [2] Sahara et al showed that SQAGs effectively inhibit the growth of implanted human lung adenocarcinoma cells, A549, in nude mice [1] Moreover, Ohta et al reported that a wide variety of cultured tumor cells were sensitive to SQAGs [6] It is very difficult to collect and purify SQAGs from natural sources but we have succeeded in the chemical synthesis of a number of forms We have found a-anomers that possess potent antitumor activ-ity but do not have many of the serious side-effects of
Keywords
mammalian DNA polymerases; MT
depolymerization activity of MCAK; SQAGs;
T7 phage display method
Correspondence
K Sakaguchi, Department of Applied
Biological Science, Tokyo University of
Science, 2641 Yamazaki, Noda, Chiba 278–
8510, Japan
Fax: +81 4 7123 9767
Tel: +81 4 7124 1501 (ext 3409)
E-mail: kengo@rs.noda.tus.ac.jp
(Received 30 October 2004, revised 20
January 2005, accepted 7 February 2005)
doi:10.1111/j.1742-4658.2005.04600.x
Sulfoquinovosylacylglycerols (SQAGs), in particular compounds with C18 fatty acid(s) on the glycerol moiety, may be clinically promising antitumor and⁄ or immunosuppressive agents They were found originally as inhibitors
of mammalian DNA polymerases However, SQAGs can arrest cultured mammalian cells not only at S phase but also at M phase, suggesting they have several molecular targets A screen for candidate target molecules using a T7 phage display method identified an amino acid sequence An homology search showed this to be a mammalian mitotic centromere-asso-ciated kinesin (MCAK), rather than a DNA polymerase Analyses showed SQAGs bound to recombinant MCAK with a KD¼ 3.1 · 10)4 to 6.2· 10)5m An in vivo microtubule depolymerization assay, using EGFP-full length MCAK fusion constructs, indicated inhibition of the micro-tubule depolymerization activity of MCAK From these results, we conclude that clinically promising SQAGs have at least two different molecular targets, DNA polymerases and MCAK It should be stressed that inhibitors of MCAK have never been reported previously so that there
is a major potential for clinical utility
Abbreviations
a-SQDG(18:0), saturated 1,2-O-diacyl-3-O-(a- D -sulfoquinovosyl)-glyceride; b-SQDG(18:0), saturated 1,2-O-diacyl-3-O-(b- D -sulfoquinovosyl)-glyceride; a-SQMG(18:0), saturated 1-O-monoacyl-3-O-(a- D -sulfoquinovosyl)-glyceride; a-SQMG(18:1), unsaturated 1-O-monoacyl-3-O-(a- D -sulfoquinovosyl)-glyceride; DMSO, dimethylsulfoxide; EGFP, enhanced green fluorescent protein; MCAK, mitotic centromere-associated kinesin; MTs, microtubules; MCAK184, His 6 -tagged MCAK truncated form (P184-G593); SQAGs, sulfoquinovosylacylglycerols.
Trang 2standard cancer chemotherapeutics [10] In contrast,
b-anomers did not show antitumorogenicity but
were toxic to lymphocytes [2] The active SQAGs are
1-O-monoacyl-3-O-(a-d-sulfoquinovosyl)-glyceride with
saturated or unsaturated fatty acids, and
O-(a-d-sulfoquinovosyl)-glyceride and
1,2-O-diacyl-3-O-(b-d-sulfoquinovosyl)-glyceride, both with saturated
fatty acids [11] The degree of inhibitory activity is
greatly dependent upon the size of the fatty acid;
SQAGs with fatty acid elements with fewer than 14
carbons do not show inhibitory effects in vitro or
in vivo [10,11] Our studies have identified possible
discrepancies with regard to mechanistic aspects of
SQAG activity in cancer cells These compounds are
considered to block replicative DNA synthesis by
sup-pressing the activity of the DNA polymerases, thus
arresting the cell cycle at S and consequently killing
the cancer cells However, aphidicolin, a
well-estab-lished DNA polymerase inhibitor with cytotoxicity
very similar to SQAGs, shows little bioactivity in vivo
As shown previously, moreover, SQAGs arrest cells
not only at the S but also at M phase [6] These
obser-vations allow us to speculate that other molecular
tar-gets may be involved in vivo, possibly inducing cell
death
T7 phage display methods are powerful and high
throughput tools for in vitro [12] and in vivo [13]
iden-tification of peptides or protein ligands In this study,
we used a T7 phage display method in combination
with immobilized biotinylated SQAG prepared on an
avidin solid phase [14–16] A sequence was thereby
identified that exhibited similarity with human mitotic
centromere-associated kinesin (MCAK) Kinesins are
molecules that convert chemical energy into physical
reactions to perform functions such as vesicle
trans-port, chromosome segregation, and organization of the
mitotic spindle Therefore, one of the other targets of
the SQAGs is probably a MCAK We show here that
SQAGs suppress microtubule depolymerization by
binding to MCAK To our knowledge, this is the first
report of an inhibitor to MCAK
Results
Screening for peptide sequences selectively
binding to SQAG in the T7 phage random
peptide library
We selected four representative SQAGs for binding
analysis:
1-O-monoacyl-3-O-(a-d-sulfoquinovosyl)-gly-ceride with saturated [a-SQMG(18:0)] or unsaturated
fatty acid [a-SQMG(18:1)];
1,2-O-diacyl-3-O-(a-d-sulfoquinovosyl)-glyceride with saturated fatty acid
[a-SQDG(18:0)]; and, 1,2-O-diacyl-3-O-(b-d-sulfo-quinovosyl)-glyceride with saturated fatty acid [b-SQDG(18:0)] (Fig 1A) Although the distribution
of a- and b-anomers in the body would be expected
to differ [10], the levels of the cytotoxicity are similar when the size of the fatty acid is the same [6] Each chemically pure compound was synthesized in our laboratory For screening peptides specifically binding
to SQAGs, 1.5· 108p.f.u per 30 lL of the T7 phage library expressed random peptide sequences was applied onto streptavidin-coated wells bearing an immobilized SQAG biotinylated derivative (Fig 1B)
We found that effective biopannning required a
Fig 1 (A) Structure of SQAGs: structure 1, R 1 ¼ CH 3 (CH 2 ) 16 CO;
R2¼ H [a-SQMG(18:0)] Structure 2, R1¼ CH 3 (CH2)7CH¼ CH(CH2)7CO; R2¼ H [a-SQMG(18 : 1)] Structure 3, R 1 ¼ R 2 ¼
CH 3 (CH 2 ) 16 CO [a-SQDG(18:0)]; Structure 4, R 3 ¼ R 4 ¼
CH3(CH2)16CO [b-SQDG(18:0)] (B) Biotinylated derivative, SQAG.
Trang 3number of rounds of elution with 1.5 m NaI followed
by washing with 0.1% Tween-20 in 100 mm Tris⁄ HCl
(pH 8.0) An illustrative example of the results of
bio-panning is shown in Fig 2 For b-SQDG(18:0), the
recovery rate of round 5 (i.e the eluted fraction of 5th
biopanning) was 7.7%, which was almost sixfold
higher than those of rounds 1–4 The DNA sequences
of 47 clones picked from round 5 were analyzed, and
finally, an oligopeptide sequence was obtained as the
clone which was highly concentrated It was composed
of 14 amino acids (NSRMRVRNATTYNS), and
here-after is called ‘clone-14’ for convenience
When the binding titer of the phage ‘clone-14’ on
the b-SQDG(18:0)-solid phase was compared to the
unselected clone (Fig 3), the recovery rate of the
for-mer was 5.1-fold higher The ‘unselected clone’ which
harbored five amino acids (NSNTR), was hardly
con-centrated in round 5 at all The data indicate that
‘clone-14’ was effectively concentrated in the
bio-panning procedure, presumably due to selective
bind-ing to the b-SQDG(18:0) molecule As indicated
below, as with the other a-anomeric SQAGs used,
a-SQMG(18:0), a-SQMG(18:1) and a-SQDG(18:0)
also bind tightly and selectively to ‘clone-14’, binding
must be unrelated to the anomeric structure (Table 1)
A homology search (fasta3) demonstrated that the
amino acid sequence of ‘clone-14’ is similar to the ‘neck
region’ of the rat, human and mouse mitotic
centro-mere-associated kinesin (MCAK) (Fig 4) [17–19]
Kine-sin family proteins generally contain the motor domain
in the N- or C-terminal of the primary sequence, and the
0
1
2
3
4
5
6
7
8
round
Fig 2 Biopanning for selecting peptide sequence bound to the
SQAG molecule The graphic indicates the process of biopanning.
A biotinylated derivative of b-SQDG(18:0) was immobilized on a
Streptavidin-coated well, and then incubated with the T7 phage
lib-rary composed of cDNA fragment inserts from Drosophila
melano-gaster In every round, unbound phages were removed by washing
three times with 100 m M Tris ⁄ HCl (pH 8.0) containing 0.1% (v ⁄ v)
Tween-20, and bound phages were eluted with 200 lL of 1.5 M
NaI at 4 C, overnight Recovery rate (%) ¼ [titer of the elute
frac-tion (p.f.u.) ⁄ titer of input (p.f.u.)] · 100 These data are shown as
the averages of two individual experiments.
Fig 3 Comparison of affinity for SQAG between the clone-14 and unselected clone Binding strengths of clone-14 and unselected clone on SQAG molecule were compared Both clones were puri-fied, amplified and adjusted the titer to 1.0 · 10 13 p.f.u.ÆmL)1 One hundred microliters of each clone were applied onto SQAG-solid phase The washing and the eluting conditions were same as those
of biopanning in Fig 2 The biotinylated SQAG did not immobilize
on the control well Increase rate of recovery for control ¼ titer of SQAG immobilized well ⁄ titer of control well.
Table 1 SPR analysis of the binding of SQAGs to the immobilized peptide, MCAK184 on a CM5 sensor chip A synthetic peptide and MCAK184 were coupled to the CM5 sensor chips Binding analy-ses of SQAGs were performed in running buffer (Experimental pro-cedures) at a flow rate of 20 lLÆmin)1at 25 C BIAEVALUATION 3.1 software was used to determine the kinetic parameters.
SQMG
KD(10)7M ) 14aa MCAK184 a-SQMG (18:0) 1700 3100 a-SQMG (18:1) 8.7 620 a-SQDG (18:0) 130 9800 b-SQDG (18:0) 15 490
A
B
Fig 4 Alignment between clone-14 amino acids sequence and human MCAK (A) Clone-14 amino acid sequence indicated similarity
to N212-S225 of human MCAK (B) The similarity site (marked by upward arrow ⁄ tripple underline) is a ‘neck region’ in MCAK This region affects the depolymerization activity of MCAK [20,29].
Trang 4position predicts the direction of walking on
micro-tubules MCAK belongs to the Kin I subfamily and its
motor domain, unlike most kinesins, is in the interior of
the protein Moreover, the protein localizes at
centro-meres, performs roles in the depolymerization of
micro-tubules, and affects chromosome segregation The neck
region is adjacent to the N-terminus of the motor
domain in MCAK From a study using a truncated
form, the neck region appears to be essential for
micro-tubule depolymerization activity Our previous data
indicated that SQAGs arrest cultured cells not only at
the S phase but also at M phase, the place in which
microtubule depolymerization occurs [6] Therefore,
SQAGs may inhibit microtubule depolymerization
activity and thereby induce cell death For this reason,
we focused on the molecular interactions between
SQAGs and the recombinant MCAK
Kinetic parameters via surface plasmon
resonance (SPR) analysis of binding between
SQAG and MCAK
The full length MCAK protein is not soluble and is
found in inclusion bodies Therefore binding between
SQAGs and a truncated version (MCAK184) was
tested using a Biosensor BIAcore instrument The
MCAK184 contains the neck region and the Kinesin
motor domain (Fig 5) Three or four different
concen-trations of each of the four SQAG [1–4] derivatives
shown in Fig 1 were employed for analyses of the
bindings to CM5 sensor chip conjugated 14aa or
MCAK184 The dissociation constants KD (m) were
determined using the biaevaluation 3.1 software
(Table 1) Values with 14aa were in the range of
KD¼ 1.3 · 10)5 to 8.7· 10)7 m, and for MCAK184
were KD¼ 3.1 · 10)4to 6.2· 10)5m
SQAG inhibits the depolymerization activity
of MCAK in vitro
To test the possibility that the interaction of SQAGs
and MCAK184 inhibited depolymerization of MTs, we
performed an in vitro depolymerization assay in the
same manner as reported previously [20–22] The
trun-cated MCAK constructed as a His6-tagged truncated
form (P184-G593; MCAK184), containing the neck
and motor domains (Fig 5A), was purified to near
homogeneity (Fig 5B) Depolymerization of the
tubu-lin polymer could be detected in SDS⁄ PAGE as an
abundance of a band of tubulin molecules released into
the supernatant The in vitro depolymerization
reac-tions contained 120 nm MCAK184 and 1500 nm of
taxol-stabilized microtubules (taxol-stabilized MTs)
MCAK184 depolymerized MTs in vitro, when incuba-ted at 24C for 30 min, but only in the presence of
1 mm ATP (Fig 6A) The presence of the ATPase inhibitor AMPPNP abolished depolymerization activity (Fig 6A) Figure 6B shows the effects of 19.6 lm a-SQMG(18:1) and 3.2 lm b-SQDG(18:0) on the depolymerization reaction The concentrations of the SQAGs used were chosen from the minimum inhibitory concentration (MIC) with MCAK184 In this case, the reaction mixture contained 2% dimethylsulfoxide (DMSO), because of the solubility of the SQAGs DMSO had no effect on the reaction (Fig 6B, upper panel) Both a-SQMG(18:1) and b-SQDG(18:0) clearly inhibited the depolymerization (Fig 6B, middle and lower panels) a-SQMG(18:0) and a-SQDG(18:0) tended also showed the same inhibition pattern (data not shown) Thus, at least under in vitro conditions, SQAGs inhibit the depolymerization activity of MCAK
by selective binding The tightness of binding may determine the degree of inhibition
SQAGs inhibit the depolymerization activity
of MCAK in vivo
To elucidate the MT-depolymerizing effects of SQAGs
in vivo, the fusion constructs of EGFP-full length MCAK were transfected into cultured cells After fix-ation, the cells were stained for tubulin and DAPI (Fig 7) and digital images were acquired using a
A
B
Fig 5 Construction and purification of MCAK184 (A) Schematic representation of the truncated form of the human MCAK struct The homology domain is a ‘clone-14’ sequence This con-struct was subcloned into pET21a vector and expression in BL21(DE3)-pLysS (B) Western blotting of MCAK 184 The crude extract was loaded onto an HiTrap Chelating HP column, then the eluted fraction was subjected to SDS ⁄ PAGE and then transfered to
a poly(vinylidene difluoride) membrane The membrane was stained with anti-His6 Ig and alkaline phosphatase A single band was present that corresponded to the molecular mass of MCAK184 (49 kDa).
Trang 5cooled CCD camera Loss of microtubule polymers
was observed in controls not treated with
a-SQMG(18:1) (Fig 7Ab, unfilled arrow), indicating that
the polymers are rapidly depolymerized However, in
the presence of a-SQMG(18:1), the polymers were not
depolymerized (Fig 7Ae)
Figure 7B shows numbers of stained cells at various
concentrations of a-SQMG(18:1) The data are from
three independent experiments The numbers of the
cells showing depolymerization of tubulin decreased
in a dose-dependent manner with increase in
a-SQMG(18:1) Similar effects were exhibited by the
other SQAGs (data not shown)
Discussion
In the present study, we have shown, using a T7 phage
display method, that mammalian mitotic
centromere-associated kinesin (MCAK) is a molecular target of
SQAGs The SQAGs inhibit the MCAK function and are likely to bind to its ‘neck region’ As this M-type kinesin is localized at centromeres and depolymerizes microtubules from their ends, an important feature of remodeling during mitosis [20–23], it is conceivable
A
B
Fig 6 Inhibition of the microtuble depolymerization activity of
MCAK184 by SQAG in vitro In all assays, 120 n M of MCAK,
1500 n M of paclitaxel stabilized microtubles, SQAGs, and reaction
components were mixed, and then were incubated at 24 C for
30 min The reaction mixture was centrifuged at 223 000 g and the
supernatant and the pellet were separated (A) MCAK184 can
depo-lymerize microtubles in vitro From left to right, paclitaxel stabilized
MTs were incubated without MCAK184, with MCAK184 and ATP,
with MCAK184 alone, and with MCAK184 and AMPPNP
Depoly-merized microtubules were visualized in the lane of the supernatant
(S), and polymerized in the lane of pellet (P) (B) SQAGs inhibited
the depolymerization of microtubles There were 2% DMSO and
ATP in all samples (Upper) MCAK 184 was incubated with 2%
DMSO; (Middle) with 19.6 l M , 4.9 l M of a-SQMG(18:1); (Lower)
with 3.2 l M , 0.8 l M of b-SQDG(18:0).
A
B
Fig 7 Inhibition on tublin depolymerization in CHO-cell transfected with EGFP-MCAK CHO cells transfected with EGFP-full length MCAK were treated with a-SQMG(18:1) (A) (a–c) Control experi-ment (d–f) Photographs of cells treated with aSQMG (18:1) (a,d) EGFP-MCAK, (b,e) staining with anti-tubulin Ig, (c,f) DAPI staining White arrow in b indicates a loss of microtubule polymer and low intensity unpolymerized tublin staining (B) The proportion of the cells with depolymerized MTs was affected by the concentration of a-SQMG(18:1) CHO cells were treated with 0 (control), 0.2, 2.3, 22.8 lM of SQAG for 24 h The ‘depolymerized cell’ on Y-axis indi-cates the number of tublin-unstained cell in 10 of EGFP-MCAK expressed cell The bar shows the standard deviation (n ¼ 5).
Trang 6that the anticancer effects of SQAGs are dependent on
the inhibition of MCAK
Although the SQAGs were found as inhibitors of
mammalian DNA polymerases [3–5, 24], the impact on
these enzymes appears too weak to explain their in vivo
anticancer activity and their weak cytotoxicity We
reported previously that SQAGs can not only arrest
cells at S phase but also at M phase [6]; thus the two
dif-ferent cell cycle phases may be impacted simultaneously
SQAGs can be separated roughly into two groups
according to the number of fatty acid molecules:
diacyl-forms (SQDGs) and monoacyl-diacyl-forms (SQMGs) Both
are sulfonic analogs of d-glucose bound with glycerol
and fatty acids Our present results showed that the
var-ious derivatives of SQAG may strongly inhibit MCAK
activity This inhibition may be independent of their
anomeric forms, as it is the case for their DNA
poly-merase inhibitor Chemical synthesis of SQMG⁄ SQDG
derivatives produces both a- and b-anomers The
b-ano-mer is not present in nature As described previously,
a-anomers of synthetic SQMG⁄ SQDG derivatives could
be potent antitumor agents without severe side-effects
In mice exposed to these agents, the immunosuppressive
effect was minor and the main visceral organs showed
no histological evidence of toxicity [10] On the other
hand, the b-anomer may be potent immunosuppressive
agents with toxic effects on lymphocytes [2] Therefore,
synthetic SQMG⁄ SQDG, chemically composed only of
carbohydrate glycerol and fatty acids, could be ideal
cancer-chemotherapeutic and⁄ or immunosuppressive
agents that could be applied clinically for longer
peri-ods The reason for tissue-specific toxicity dependent on
the different configuration can be explained by the
inhibition of neither DNA polymerases nor MCAK,
pointing to the possibility of further molecular targets
As DNA polymerases are essential for DNA
replica-tion and repair, their inhibireplica-tion will induce cell cycle
arrest at the S phase The MIC ranges for DNA
polymerases in vitro were low at 1–50 lm [6,11], while
cytotoxicity was evident at 50–100 lm Inhibition of
MCAK activity occurred at 0.8–20 lm Therefore, cell
death in vitro may occur as a result of synergistic
actions SQAGs are also known to act against
inflam-mation [25], respiration of spermatozoa [26–28],
HIV-RT (human immunodeficiency virus-reverse
tran-scriptase) [4,5,7], AIDS virus [4], the P-selectin receptor
[8] and a-glucosidase [29] With the exception of the
last two, the in vivo molecular targets for these effects
have yet to be elucidated Interestingly, the binding
analysis of SQAGs and MCAK184 showed that the
kinetic constant (KD) for the interaction between
a-SQMG(18:1) and MCAK184 was lowest recorded
(6.2· 10)5m) (Table 1) Of the SQAGs used here,
a-SQMG(18:1) is the strongest anticancer agent [10], suggesting that the tightness of binding to MCAK is important for in vivo bioactivity As described previ-ously, the inhibition of DNA polymerases occurs by tight binding to molecular pockets on their surfaces The degree of inhibition depends on the KD (m) between the SQAG and the enzyme
Although there are many drugs that bind tubulin directly (such as paclitaxel or nocodazole, which
over-or understabilize microtubules, respectively), a drug that targets the M-type kinesin has never been reported Thus the SQAGs may be of particular significance, not only with regard to their clinical applications, but also for analysis of the functions of MCAK
Experimental procedures
SQAGs
SQAGs and a biotinylated derivative of SQAG were syn-thesized in our laboratory (Fig 1) [3–5,14]
Construction of a T7 phage library from Drosophila melanogaster
Random primers, 5¢-methylated dCTP, T4 DNA poly-merase, T4 ligase, EcoRI⁄ HindIII linkers, EcoRI, HindIII, T7Select10–3b vector, and T7 packaging extracts were pur-chased from Novagen (Darmstadt, Germany) [15] Con-struction of the phage library was carried out according to the manufacturer’s instructions In brief, aliquots (80 lg) of total RNA, extracted from D melanogaster Kc cells, (pro-vided by M Yamaguchi, Kyoto Institute of Technology, Japan) were used to construct a cDNA library Total RNA was treated with Oligotex-dT30 super (Takara, Shiga, Japan) to produce poly(A)+ RNA suitable for random primed cDNA synthesis cDNA synthesis was performed using 4 lg of poly(A)+ RNA 5¢-Methylated dCTP was then incorporated into both strands, without extraction or precipitation between the first and second strand synthesis The cDNA was then treated with T4 DNA polymerase to generate flush ends and ligated with directional EcoRI⁄ Hin-dIII linkers Following linker ligation, the cDNA was diges-ted sequentially with EcoRI and HindIII, then inserdiges-ted into EcoRI⁄ HindIII digested T7Select10–3b vector arms The cDNA was cloned into the EcoRI⁄ HindIII site of the T7 phage 10–3b vector and packaged into phage [15] The titer
of this library was 1.6· 1010p.f.u.ÆmL)1
T7 phage clone biopanning procedure and DNA sequence analysis
A biotinylated derivative of SQAG was immobilized on a Streptavidin-coated ELISA plate (Nalge Nunc International,
Trang 7Wiesbaden, Germany) overnight at 4C Unbound SQAG
was removed by washing three times with 150 lL Tris
buf-fer (100 mm Tris⁄ HCl, pH 8.0) and plates were blocked
with 200 lL of Tris buffer containing 3% (w⁄ v) BSA for
1 h The plates were washed three times with 200 lL of
Tris buffer and then incubated, for 3 h with gentle rotation,
with a T7 phage library composed of cDNA fragment
inserts from D melanogaster Unbound phage was removed
by washing three times with 0.1% (v⁄ v) Tween-20 in Tris
buffer Bound phage was eluted from each plate by first an
overnight incubation at 4C with 200 lL of 1.5 m NaI,
and then four washes with 100 lL of 1.5 m NaI The
super-natant (total 600 lL) from both steps was collected and
regarded as the eluted fraction An aliquot (10 lL) was
used to determine the titer of detached phage at each round
of selection The remainder was amplified by the plate
ly-sate amplification method [15] for a new round of selection
in the same manner as described above
Following five rounds of selection, 47 plaques were
arbitrarily picked up from LB plates and each dissolved in
phage extraction buffer (20 mm Tris⁄ HCl, pH 8.0, 100 mm
NaCl, 6 mm MgSO4) In order to disrupt the phages, the
extract was heated at 65C for 10 min Phage DNA was
then amplified by PCR, using T7 SelectUP and T7
Select-DOWN primers (T7Select Cloning kit, Novagen) PCR
products were cloned in the pGEM-T vector (Promega,
Sequencer 4200S (Aloka, Tokyo, Japan) From these
sequence results, the amino acid sequence displayed on the
T7 phage capsid was determined A homology search
(fasta3) demonstrated that the amino acid sequence of
clone-14 is similar to the neck region of the MCAK
Some parameters were changed: Database, SwissProt;
Expectation upper value, 50; Matrix, BL50; Number of
alignments, 50
Comparisons of affinity for SQAG with selected
and unselected T7 phage single clones
The affinity of the candidate clone (positive) for SQAG was
compared with that of an unselected clone (negative) The
negative clone showed low selectivity with biopanning
Both single clone phages were amplified for liquid lysate
amplification [15] and adjusted to a titer of 1.0· 1013
p.f.u.ÆmL)1 One hundred microliters of each single phage
suspension (i.e 1012p.f.u.) was applied onto SQAG
immo-bilized plates Washing and eluting proceeded as described
above for biopanning The titer of the eluted fraction was
determined
Construction of recombinant human MCAK
MCAK cDNA was derived from a human peripheral blood
cDNA library by PCR (forward primer: 5¢-ATGGC
CATGGACTCGTCGCT-3¢, reverse primer; 5¢-TCACTG
GGGCCGTTTCTTGC-3¢) The neck and motor domains
of MCAK cDNA (550–1770), conjugated with NdeI and XhoI restriction sites, were cloned into the pET21a expres-sion vector (Novagen) EGFP-full length MCAK was made
by the cloning of XhoI-BamHI MCAK cDNA fragment (forward primer: 5¢-CTCGAGATGGCCATGGACTCGT CG-3¢, reverse primer: 5¢-GGATCCTCACTGGGGCCGTT TCTT-3¢) into the pEGFP-C3 vector (BD Biosciences, Tokyo, Japan)
His6-tagged MCAK184 protein preparation
MCAK184 protein was overexpressed, purified for SPR analysis, and an in vitro MT depolymerization assay was conducted Protein expression was performed by transform-ing the construct into BL21 (DE3)-pLysS (Novagen) and growing these bacteria in 1 L of Luria–Bertani medium containing 50 lgÆmL)1 of kanamycin, 100 lgÆmL)1 chlo-ramphenicol Cells were grown and treated with 1 mm of isopropyl thio-b-d-galactoside After 3 h, they were harves-ted by centrifugation at 3000 g for 15 min Cell pellets (3.5 g) were resuspended in 30 mL of ice-cold column bind-ing buffer (20 mm sodium phosphate, pH 7.4, 0.5 m NaCl,
35 mm imidazole) and sonicated Cell lysates were centri-fuged at 27000 g for 20 min and the soluble protein frac-tion was collected as a crude extract and loaded onto a
5 mL HisTrap HP column (Amersham Biosciences, Foster City, CA, USA) of the FPLC system (A¨KTA 1 explorer, Amersham Biosciences) with a flow rate of 1 mLÆmin)1 The column was washed firstly with 100 mL binding buffer and then washed with 20 mL of buffer (20 mm sodium phosphate, pH 7.4, 0.5 m NaCl, 65 mm imidazole) Finally, MCAK184 was eluted with 100 mL of eluting buffer (20 mm sodium phosphate, pH 7.4, 0.5 m NaCl, 270 mm imidazole) For surface plasmon resonance (SPR) analysis, the eluted MCAK184 protein was dialyzed against HBS⁄ EP buffer [10 mm Hepes, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% (v⁄ v) Tween-20] For the in vitro MT depolymeriza-tion assay, the eluted MCAK184 protein was dialyzed against sodium phosphate buffer (50 mm sodium phos-phate, pH 7.0, 150 mm NaCl)
Surface plasmon resonance (SPR) analysis
The binding characteristics of SQAGs and a synthetic peptide ‘NSRMRVRNATTYNS’ (ANYGEN, Gwang-ju, Korea), MCAK184, was analyzed using a Biosensor 3000 instrument (BIAcore AB, Uppsala, Sweden) with CM5 research grade sensor chips (BIAcore) The synthetic pep-tide (332 lgÆmL)1, 170 lL) in coupling buffer (10 mm sodium carbonate⁄ sodium hydrogen carbonate, pH 8.5) was injected over a CM5 sensor chip at a 10 lLÆmin)1 of flow rate to capture the peptide on the carboxymethyl dex-tran matrix of the chip by using amine coupling at 25C The surface was activated by injecting a solution containing
Trang 80.2 m N-ethyl-N¢-dimethylaminopropyl carbodiimide (EDC)
and 50 mm N-hydroxysuccimide (NHS) for 14 min The
peptide was injected and the surface was then blocked by
injecting 1 m ethanolamine at pH 8.5 for 14 min This
reac-tion immobilized about 1500 resonance units (RU) of the
peptide When MCAK184 (332 lgÆmL)1, 170 lL) in
coup-ling buffer (10 mm acetic acid⁄ sodium acetate, pH 4) was
injected, about 2700 RU were immobilized Binding
analy-sis of SQAGs was performed in running buffer [10 mm
Hepes, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% (v⁄ v)
Tween-20, 8% (v⁄ v) DMSO] at a flow rate of 20 lLÆmin)1
at 25C To measure the binding specificity and kinetics
for 14 amino acids (aa), various SQAGs were injected for
120 s [a-SQMG(18:0): 22.8, 91, 364, 728 lm; a-SQMG
(18:1): 0.2, 1.14, 2.28, 5.46 mm; a-SQDG(18:0): 60, 118,
236, 472 lm; b-SQDG(18:0): 7.4, 17.6, 23.5, 29.4 lm] To
measure MCAK184, various SQAGs were injected for
120 s [a-SQMG(18:0): 91, 364, 728 lm; a-SQMG(18:1):
204, 319, 364 lm; a-SQDG(18:0): 118, 235, 470 lm;
b-SQDG(18:0): 70.6, 76.4, 82.3 lm] Association and
disso-ciation were each measured for 120 s at 20 lLÆmin)1
biaevaluation 3.1 software (BIAcore) was used to
determine the kinetic parameters
In vitro microtubule depolymerization assay
A microtubule depolymerization assay using polymerized,
taxol-stabilized tubulin from bovine cytoskeleton (Denver,
CO, USA), was performed as described previously [20–22]
For the assay shown in Fig 6A, 120 nm MCAK184 in
20 lL of column eluting buffer (250 mm imidazole, pH 7.0,
300 mm KCl, 0.2 mm MgCl2, 0.01 mm Mg-ATP, 1 mm
dithiothreitol and 20% glycerol) was mixed with 1500 nm
taxol-stabilized microtubules in 80 lL of BRB80 (80 mm
Pipes, pH 6.8, 1 mm EGTA, and 1 mm MgCl2), 12.5 lm
taxol, 1 mm dithiothreitol, and 1.25 mm Mg-ATP⁄ 1.25 mm
Mg-adenyl-5¢-yl imidodisphosphate incubated at 24 C for
30 min, and then centrifuged at 223 000 g for 15 min For
the assay shown in Fig 6B, there was 2% (final
concentra-tion) DMSO in all samples Supernatants and pellets were
assayed for the presence of tubulin on Coomassie-stained
SDS⁄ polyacrylamide gels
Cell transfection and immunofluorescence
CHO-K1 cells (Japan Health Sciences Foundation, Tokyo,
Japan) were grown in Ham’s F12 medium with 10% (v⁄ v)
fetal bovine serum Transfection of pEGFP-C3-full length
MCAK was performed with LipofectamineTM2000
(Invitro-gen, Carlsbad, CA, USA) After transfection, cells were
cul-tured for 24 h and various concentrations of SQAGs were
administered Cells were exposed for 24 h, fixed with 1%
paraformaldehyde in cold methanol for 10 min, then
incu-bated for 1 h with a mouse anti-(a-tubulin DM1A IgG) Ig
(Sigma-Aldrich, St Louis, MO, USA) at 1 : 1000 dilution
and a rhodamine-conjugated anti-mouse Ig (Chemicon International, Temecula, CA, USA) at 1 : 100 dilution in NaCl⁄ Pi, 0.1% (v⁄ v) Triton X-100 and 1% (w ⁄ v) BSA for
1 h Finally they were washed with NaCl⁄ Pi and mounted
in mounting medium [NaCl⁄ Pi, 4¢,6-diamidino-2-phenyl-indole (DAPI), 10% (v⁄ v) glycerol] for analysis under a microscope (Axioskop 2 plus, Zeiss, Tokyo, Japan) Digital images were acquired with a cooled CCD camera (Axio-Cam HRm, ZEISS) controlled by axiovision 3.0 software (Zeiss)
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