Mammalian mitotic centromere-associated kinesin (MCAK) A new molecular target of sulfoquinovosylacylglycerols novel antitumor and immunosuppressive agents Satoko Aoki 1,2 , Keisuke Ohta 1 , Takayuki Yamazaki 1 , Fumio Sugawara 1,2 and Kengo Sakaguchi 1,2 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 K D ¼ 3.1 · 10 )4 to 6.2 · 10 )5 m.Anin 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. 2132 FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS standard 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 1,2-O-diacyl- 3-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 · 10 8 p.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; R 2 ¼ H[a-SQMG(18:0)]. Structure 2, R 1 ¼ CH 3 (CH 2 ) 7 CH¼ CH(CH 2 ) 7 CO; R 2 ¼ 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 CH 3 (CH 2 ) 16 CO [b-SQDG(18:0)]. (B) Biotinylated derivative, SQAG. S. Aoki et al. A molecular target of SQAGs FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS 2133 number 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 12345 round Recovery rate (%) 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 )1 at 25 °C. BIAEVALUATION 3.1 software was used to determine the kinetic parameters. SQMG K D (10 )7 M) 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]. A molecular target of SQAGs S. Aoki et al. 2134 FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS position 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 K D (m) were determined using the biaevaluation 3.1 software (Table 1). Values with 14aa were in the range of K D ¼ 1.3 · 10 )5 to 8.7 · 10 )7 m, and for MCAK184 were K D ¼ 3.1 · 10 )4 to 6.2 · 10 )5 m. 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 His 6 -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 24 °C for 30 min, but only in the presence of 1mm 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 con- 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-His 6 Ig and alkaline phosphatase. A single band was present that corresponded to the molecular mass of MCAK184 (49 kDa). S. Aoki et al. A molecular target of SQAGs FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS 2135 cooled 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,4.9lM of a-SQMG(18:1); (Lower) with 3.2 l M,0.8lM 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). A molecular target of SQAGs S. Aoki et al. 2136 FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS that 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-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 inhibition 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 (K D ) for the interaction between a-SQMG(18:1) and MCAK184 was lowest recorded (6.2 · 10 )5 m) (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 K D (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 inserted 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 · 10 10 p.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, S. Aoki et al. A molecular target of SQAGs FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS 2137 Wiesbaden, Germany) overnight at 4 °C. 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 lLof 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 4 °C 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 MgSO 4 ). In order to disrupt the phages, the extract was heated at 65 °C 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, Madison, WI, USA) and sequenced using a DNA 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 · 10 13 p.f.u.ÆmL )1 . One hundred microliters of each single phage suspension (i.e. 10 12 p.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). His 6 -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 mL Æmin )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 25 °C. The surface was activated by injecting a solution containing A molecular target of SQAGs S. Aoki et al. 2138 FEBS Journal 272 (2005) 2132–2140 ª 2005 FEBS 0.2 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 25 °C. 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 l m; 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 MgCl 2 , 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 MgCl 2 ), 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 Lipofectamine TM 2000 (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 ⁄ P i , 0.1% (v ⁄ v) Triton X-100 and 1% (w ⁄ v) BSA for 1 h. 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