HSET belongs to kinesin family (Hirokawa and Takemura, 2005) and most of kinesin proteins share similar sequences of amino acid with HSET (Fig. 1). Amino acid sequence and each domain of HSET are shown in Figure 2. To increase specificity of Ab to HSET, it is essential to avoid targeting the regions which show high homology with other kinesins. We performed BLAST search for HSET to avoid cross-reactivity of anti-HSET Ab with kinesin proteins (Fig. 3). N-terminus (Met1 - Arg134) and C-terminus (Asn626 - Arg672) regions of HSET showed relatively low homology to other kinesins, compared to motor region of HSET.
The coiled coil region (Glu249 - Glu306) was considered to be unsuitable to use as antigen because it has many hydrophobic amino acids and moderate similarity with other kinesins.
Finally we designed 12 amino acid-length peptides from 14 hydrophilic regions corresponding to N- and C-terminus of HSET for mice immunization (Table 2) and the location of each peptide in HSET was indicated in Table 3.
10
Figure 1. Structures of kinesins. The structures of the kinesin family. The motor domains (pink-colored), the ATP-binding site (a thin purple line), the microtubule-binding site (a thick purple line), the dimerization domains (yellow stripes), the forkhead-associated regions (red stripes) and pleckstrin homology domains (orange stripes) are shown as schematic diagram. The number of amino acids of each molecule is shown on the right. A partial region (1st - 609th amino acids) of KIFC1 (HSET) is compared to other kinesin family.
11
10 20 30 40 50 60 MDPQRSPLLE VKGNIELKRP LIKAPSQLPL SGSRLKRRPD QMEDGLEPEK KRTRGLGATT
70 80 90 100 110 120 KITTSHPRVP SLTTVPQTQG QTTAQKVSKK TGPRCSTAIA TGLKNQKPVP AVPVQKSGTS
130 140 150 160 170 180 GVPPMAGGKK PSKRPAWDLK GQLCDLNAEL KRCRERTQTL DQENQQLQDQ LRDAQQQVKA
190 200 210 220 230 240 LGTERTTLEG HLAKVQAQAE QGQQELKNLR ACVLELEERL STQEGLVQEL QKKQVELQEE
250 260 270 280 290 300 RRGLMSQLEE KERRLQTSEA ALSSSQAEVA SLRQETVAQA ALLTEREERL HGLEMERRRL
310 320 330 340 350 360 HNQLQELKGN IRVFCRVRPV LPGEPTPPPG LLLFPSGPGG PSDPPTRLSL SRSDERRGTL 370 380 390 400 410 420 SGAPAPPTRH DFSFDRVFPP GSGQDEVFEE IAMLVQSALD GYPVCIFAYG QTGSGKTFTM 430 440 450 460 470 480 EGGPGGDPQL EGLIPRALRH LFSVAQELSG QGWTYSFVAS YVEIYNETVR DLLATGTRKG 490 500 510 520 530 540 QGGECEIRRA GPGSEELTVT NARYVPVSCE KEVDALLHLA RQNRAVARTA QNERSSRSHS 550 560 570 580 590 600 VFQLQISGEH SSRGLQCGAP LSLVDLAGSE RLDPGLALGP GERERLRETQ AINSSLSTLG 610 620 630 640 650 660 LVIMALSNKE SHVPYRNSKL TYLLQNSLGG SAKMLMFVNI SPLEENVSES LNSLRFASKV 670
NQCVIGTAQA NRK
Figure 2. Amino acids sequence of human HSET. Amino acid from 36 to 52th (orange-colored) is a nuclear localization signal which makes HSET localizing at nuclear. Coiled coil region (142-306th amino acid, pink-colored) are related to dimerization of HSET. Motor domain corresponding to 307-594th amino acid (blue-colored) has nucleotide binding site (410-417th amino acid, cyan-colored) and ATP hydrolyzing activity. Underlined italic letters indicate helix structure.
12
Figure 3. BLAST search of whole HSET protein (Met1 - Lys673) sequence. Whole HSET protein sequence was analyzed using BLAST to know which region has similarity with other kinesin proteins. The red bar on the top indicates whole HSET protein against in which sequence similarity with other proteins in the database was compared.
13
Table 2. Peptides sequence of HSET used for immunization
No. Peptide sequence
(12 amino acid-length) Amino acid number
1 PPMAGGKKPSKR 123-134
2 PSLTTVPQTQGQ 70-81
3 QKSGTSGVPPMA 115-126
4 SRLKRRPDQMED 33-44
5 KPVPAVPVQKSG 107-118
6 EDGLEPEKKRTR 43-54
7 KAPSQLPLSGSR 23-34
8 MDPQRSPLLEVK 1-12
9 KGNIELKRPLIK 12-23
10 IATGLKNQKPVP 99-110
11 RTRGLGATTKIT 52-63
12 NSLGGSAKMLMF 626-637
13 AKMLMFVNISPL 632-643
14 NQCVIGTAQANR 661-672
14
Table 3. The location of peptide antigens used for immunization
15 protein Aga2 using yeast expression vector and displayed on the surface of yeast cells (Fig. 4A).
The expression level of HSET fragment displayed on the yeast was detected using c-myc tag located at C-terminus of HSET and FITC conjugated secondary Ab through flow cytometry (Fig.
4B). As a result, more than 60% of yeast cells successfully displayed HSET fragment. To use yeast surface displayed protein as an antigen of ELISA, we determined whether yeast cells could be coated on the 96-well microtiter plate or not. As a result, it is confirmed that yeast cells could be coated on the plate and detected by anti-HSET Ab (Fig. 4C).
To ascertain that mAbs bind to full-length protein, whole HSET protein tagged with hexa-histidine at the C-terminus was purified from E.coli. Protein in cell lysates was captured by cobalt resin via histidine tag and eluted by addition of high concentration imidazole solution.
We obtained 700 µg of protein per 1 L culture. The purity of protein was confirmed by SDS-PAGE and western blotting (Fig. 5). The bands below 50 kDa detected by western blotting were thought to be the degraded forms of HSET. The band near 70 kDa (Fig. 5A) seems to be HSET protein in which N-terminus and C-terminus are lost, because it was not detected by both anti-His and anti-HSET (Met1 - Lys50) mAbs in western blotting (Fig. 5B and 5C).
16 A
B C
Figure 4. Confirmation of yeast surface displayed HSET fragment. (A) Schematic diagram indicates the structure of yeast displayed protein. HSET antigen (Met1 - Ala100) and aga2 protein were linked through (Gly4Ser)3 linker. HA tag and c-myc tag were located at the end of aga2 and antigen, respectively. (B) Yeast displayed Aga2-HSET fusion protein are monitored by flow cytometry. Using anti-myc mAbs and FITC conjugated secondary Ab, it is found that more than 60% of yeast cells were expressing HSET fragment. (C) HSET fragment displaying yeast cells were coated on the 96-well plate and detected by HSET specific mAbs through
17
A B C
Figure 5. Purification of whole HSET (Met1 - Lys673) protein. (A) SDS-PAGE of the whole HSET protein (74 kDa) purified by affinity chromatography using a cobalt resin. Protein (10 μg) were loaded on 10% acrylamide gels under denature condition and visualized with Coomassie Blue. (B, C) Western blottings. The purified whole HSET protein was detected with either mouse anti-His tag mAb (B) or mouse anti-HSET (the region of Met1 - Lys50) mAb (C), followed by subsequent AP-goat anti-mouse IgG.
18
C. Binding of anti-HSET mAbs to different forms of HSET antigen
Twenty-eight hybridoma cell lines were generated using a hybridoma technique with immunization of HSET peptides (Table 2). To get high concentration of mAb, we injected hybridoma cells into abdominal cavity of mice and collected the Ab-rich ascitic fluids. On the other hands, Abs were purified from the supernatant of hybridoma cells by affinity chromatography. The ascitic fluids and the Abs purified from hybridoma culture supernatant were analyzed by western blotting and SDS-PAGE (Fig. 6 and 10).
Four different forms of HSET antigens were prepared to analyze the binding activity of twenty-eight anti-HSET mAbs. Three antigens including 12 amino acid-length peptides used for mice immunization, 50 amino acid-length synthetic peptide (Met1 - Lys50), and 100 amino acid-length HSET fragment (Met1 - Ala100) displayed on the yeast surface were used for ELISA to analyze the binding activities of mAbs in ascitic fluids. The purified whole HSET antigen was used to analyze the binding of the purified IgG mAbs.
First, we analyzed the binding of mAbs in ascitic fluids to 12 amino acid-length peptides.
Most of clones bound to each peptide which had been used for immunization with high extent, except for clone 1C272, 2C279, 2C280, and 2C281 (Fig. 7). To confirm whether mAbs in ascitic fluids bind to longer synthetic peptide (Met1 - Lys50), ELISA was performed. Anti-HSET mAbs in ascitic fluids were incubated with the peptide-coated wells and then detected with AP-conjugated anti-mouse IgG Ab. Clone 6C407, 8C346, 9C352, and 9C353 showed high binding, compare to positive control that commercial anti-HSET mAb (Fig. 8). MAbs in ascitic fluids, which target epitopes between Met1 - Ala100 of HSET, were analyzed by ELISA using the yeast displayed HSET fragment antigen (Met1 - Ala100). Clone 8C346 showed relatively
19
high binding and clone 6C407 and 7C315 bound 1.7-fold greater than negative control to HSET N-terminus (Met1 - Ala100) displayed on yeast cells (Fig. 9).
In ELISA using ascitic fluids, 2 µl of ascitic fluids was treated per well. Considering that the amount of mAbs in 3 µl of ascitic fluids was similar to 1 µg of positive control mouse IgG (Fig. 6), about 1 µg of mAbs were treated to each well. Although concentration of mAbs was different between clones, it seems that binding of mAbs to antigen was mainly determined by the affinity of mAbs. It was because that 8C346 Ab amount in ascitic fluids was smaller than 8C344, but 8C346 showed higher binding to 50 amino acid-length peptide and yeast displayed HSET fragment in ELISA (Fig. 8 and 9).
When the purified mAbs were stored in 50% of glycerol/ 0.05% of sodium azide, aggregation of the purified mAbs was observed, making us unable to determine accurate concentration of the purified mAbs. The concentration of the purified mAbs used for ELISA was compensated based on SDS-PAGE result (Fig. 10). The binding activity of the purified mAbs to whole HSET (Met1 - Lys673) protein was confirmed by ELISA. Clone 1C274, 2C280, 2C281, 6C407, 9C352, and 9C353 bound to whole HSET protein, to similar extent with a positive control (Fig. 11).
20
Figure 6. Western blotting of the ascitic fluids containing anti-HSET mAbs. The aliquots (3 µl) of ascitic fluids that had been obtained by i.p. injection of the hybrioma cells were run on 12% acrylamide gels under denature condition. Then, the mAbs were detected by Western blotting using AP-conjugated goat anti-mouse IgG (Fc-specific). Pure mouse IgG of 1µg was loaded as a positive control to compare the concentration of mAbs in ascitic fluids.
21
1C272 1C274 1C275 2C279 2C280 2C281 3C288 3C289 3C290 3C292 4C295 4C298 5C303 5C304 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 10C358 10C362 11C308 N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Immunogen peptide coating PBS coating
Hybridoma clone
Abs at 405 nm
Figure 7. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to peptide used for immunization. Eleven kinds of immunogen peptides (0.05 μg/well) were coated on the 96-well plate, followed by treatment of 50-fold diluted ascitic fluids. MAbs that bound to immunogen were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
22
4C295 4C298 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 P.C N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 8. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to synthetic HSET peptide (Met1 - Lys50). Synthetic HSET peptide (1 µg/ml) was coated on 96-well plate and then 50-fold diluted ascitic fluids were treated. MAbs were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used.
Commercial anti-HSET mAb (from Abcam) was used as a positive control. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
23
2C279 2C280 2C281 4C295 4C298 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 11C308 P.C N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 9. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to yeast displayed HSET fragment (Met1 - Ala100). HSET fragment displaying yeast cells (5 × 105 cells/well) were coated on 96-well plate and then 50-fold diluted ascitic fluids were treated. The bound mAbs were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used. Commercial anti-HSET mAb (from Abcam) was used as a positive control. The first digit of the clone name stands for peptide antigen number.
Data represent the mean ± S.D. of triplicate wells.
24
Figure 10. SDS-PAGE of the purified anti-HSET mAbs. Anti-HSET mAbs collected from hybridoma cell culture supernatant were purified using protein G column. About 20 µg of each protein was loaded on 12% acrylamiade gels and visualized with coomassie blue. In the reducing condition, heavy chain and light chain protein are localized near 50 kDa and 25 kDa size respectively.
25
1C272 1C274 1C275 2C279 2C280 2C281 3C288 3C289 3C290 3C292 4C295 4C298 5C303 5C304 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 10C358 10C362 11C380 anti His Ab anti HSET 1-100 Ab anti HSET 625-673 Ab mouse IgG
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 11. ELISA for the binding of the purified anti-HSET mAbs to whole HSET protein antigen. The anti-HSET mAbs (5 µg) purified from culture supernatants were added to the wells coated with whole HSET (Met1 - Lys673) protein (2.5 µg). The bound IgG mAbs were detected using AP-conjugated anti-mouse Ab. Irrelevant polyclonal mouse IgG was used as a negative control. As positive controles, commercial mAbs against N-terminus (1-50 aa) and C-terminus (625-673 aa) of HSET, and His tag were used as positive controls. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
26
D. Comparison of binding activity between mAbs
We summarized the results of ELISA of Fig. 7, 8, 9, and 11 (Table 4). Clone 6C407 and 8C346 against Glu43 - Arg54 and Met1 - Lys12 of HSET, bound all kinds of antigen and were expected to have high binding activity. Clone 2C280 and 2C281, these bound to whole HSET protein, but not to yeast displayed protein. There are two clones that could bind peptide and whole HSET protein but yeast displayed protein, clone 9C352 and 9C353 which have epitope between Lys12 - Lys23. Because of different targeting region of clone 1C274 and 3C290 (Pro123 - Arg134 and Gln115 - Ala126, respectively), they were not available using Met1 - Lys50 peptide and Met1 - Ala100 yeast displayed protein as antigens to perform ELISA.
Nevertheless, clone 1C274 and 3C290 showed comparatively high binding to whole HSET protein. All of these clones were targeting N-terminus of HSET, not C-terminus. The reason could be explained when amino acid sequence of human HSET and mouse KIFC1, were aligned.
Because C-terminus region of HSET and KIFC1 were almost similar sequence, it is thought that C-terminus of HSET could not induce immune response in mouse (Fig 12).
27 Table 4. Summary of anti-HSET mAbs
MAbs in ascitic fluids Purified mAbs Peptide divided by negative control value (1-1.5: +, 1.5-2: ++, 2-2.5: +++, 2.5-3:
++++, >3: +++++).
28
Figure 12. Sequence comparison between human HSET and mouse KIFC1. Human HSET and its mouse homolog, KIFC1, have higher homology of amino acid sequence at C-terminus than N-terminus.
29 E. Sequence analysis of anti-HSET mAbs
Sequence of mAbs were analyzed for the generation or enginieering of recombinant Ab.
DNA sequences of all mAbs were analyzed regardless of their binding activities. It is because that even mAbs with low binding activity against the prepared HSET could strongly bind to the intracellularly expressed HSET. Variable region genes were amplified and read by the primers that hybridize to immunoglobulin genes (Table 1). In case of clone 5C303 and 6C406, Vĸ genes were not able to be identified because those genes were amplified by MKV2 primer which also annealed to pseudo Vĸ gene from myeloma fusion partner cell. In case of clone 8C342, VH gene was not amplified by any MHV primers for unknown reason.
Comparing the sequences of mAbs, mAbs that target same region of HSET had similar variable region sequence (Table 5). Variable heavy chain genes (VH) of 3C288, 3C289, 3C290, and 3C292 that target Gln115 - Ala126 (peptide 3) were 100% identical. In particular, 3C288 and 3C289 had 100% identical variable kappa chain (Vĸ). Clone 6C404 and 6C406 that target Glu43 - Arg54 (peptide 6) had 100% identical VH sequence, although their Vĸ sequences could not be compared. Clone 4C295 and 4C298 that recognize Ser33 - Asp44 (peptide 4) also had 100% identical Vĸ and were different in only one amino acid on VH.
30
Table 5. Amino acids sequence of variable regions in anti-HSET mAbs
Variable heavy chain
31 Variable kappa chain
32
IV. DISCUSSION
It is well known that several cancer cells have amplified centrosomes compare with normal cells. There were many attempts to target centrosome bundling mechanism as a development of therapeutic molecules, including antifungal drug griseofulvin (Rebacz et al., 2007), kinesin spindle protein inhibitor, CENP-E inhibitor (Huszar et al., 2009), and phenanthrene-derived PARP inhibitor (Castiel et al., 2011). In recent studies, some small molecules that have been developed to block the centrosome clustering function of HSET induced the death of cancer cells, suggesting that HSET would be a promising target molecule for cancer therapy (Watts et al., 2013; Wu et al., 2013).
Generally, comparing to small molecule inhibitors which can cause side effects from the lack of specificity, mAbs are known to have high specificity against target antigen (Huang et al., 2004). Moreover, the half-lives of mAbs are 4-fold longer than small molecule inhibitors (Dancey and Sausville, 2003).
In this study, we successfully produced mAbs against N-terminus region of HSET molecules using a hybridoma technique, with the expectation to inhibit the function of HSET in the cancer cells in which function of HSET is indispensable for their cell division. HSET sequences were analyzed by BLAST search to design antigen for immunization. Some regions of HSET which show relatively hydrophilic and low homology with other proteins were synthesized as 12 amino acid-length peptides. The advantage of short length antigens, instead of immunization with full length protein, is that the production of anti-HSET mAbs which show cross-reactivity with other kinesin could be avoided. Though several mAbs to peptide or whole protein form of HSET were obtained, but the binding activity against endogenous HSET
33
molecule through intracelluar expression of these mAbs should be confirmed by the method of immunoprecipitation and immunofluorescence.
Binding activity of mAbs was different, depending on the types of antigen. It may be two reasons as follows. First, the glycosylation pattern may affect the binding site of mAbs. HSET fragment displayed on the yeast surface can be glycosylated in unpredicted regions of protein.
Different glycosylation pattern can lead to the change of protein conformation. Second, mAbs that had been derived from the immunization with HSET peptide antigens could not recognize the secondary/tertiary structure of intact HSET protein.
In fact, we tried simultaneously a hybridoma technique and a phage display technique to obtain anti-HSET mAbs. Unfortunately, however, none of the mAbs was obtained from phage display technique in which phage surface-displayed human Ab (single chain variable fragments) library was panned against the peptide antigens (corresponding to Met1 - Ala100 and Gln625 - Lys673 of HSET, respectively) as well as yeast surface-displayed HSET fragment (Met1 - Ala100).
All most of IgG mAbs of 150 kDa cannot enter cells. To target intracellular molecules using an Ab, internalization of Ab should be allowed. As a matter of fact, many researchers pay attention to intracellular targeting Ab and a variety of attempts are being tried to penetrate Ab into the cells (Kaiser et al., 2014). HSET is localized in nucleus and cytosol during interphase and mitosis (Cai et al., 2009), respectively. It implies that anti-HSET Ab should enter across the cellular membrane to target HSET molecule. Therefore, to inhibit HSET function by the treatment of anti-HSET mAbs to cancer cells, the conversion of non-internalizing anti-HSET mAbs to the internalizing Ab (intrabody) by Ab engineering should be followed.
34
V. CONCLUSION
We generated mouse mAbs against HSET protein involved in centrosome clustering essential for division of the cancer cells with extra-centrosome, and analyzed the binding activity of the mAbs to HSET.
35
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