JP2003235550A - SULFATED LEWIS x ANTIGEN PRODUCING CELL AND SULFATED LEWIS a ANTIGEN PRODUCING CELL - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide cells useful for effective screening of medicine useful for antitumor agent or metastasis control agent. <P>SOLUTION: The invention provides a sulfated Lewis x antigen producing cell or a sulfated Lewis a antigen producing cell having sulfated Lewis x antigen and sulfated Lewis a antigen productivity imparted the productivity of the sulfated Lewis x antigen producing cell and the sulfated Lewis antigen producing cell by transducing at least a kind of gene of the enzyme which is necessary for sulfation to a host cell having a deletion of enzyme necessary for sulfation of a Lewis x antigen of Lewis a antigen. The sulfated Lewis x antigen producing cell and sulfated Lewis a antigen producing cell are useful for effective screening of medicine useful for antitumor agent or metastasis control agent are firstly provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Lewis antigen-producing cell genetically engineered with a Lewis antigen-producing ability. The cell of the present invention can be used for screening an anticancer agent or a cancer metastasis inhibitor.

[0002]

2. Description of the Related Art Conventionally, the screening of drugs for treating diseases has generally been carried out by administering a candidate drug to a model animal suffering from the disease or model cells having a lesion due to the disease to obtain a disease state or lesion. It depends on whether or not there is any improvement.

However, according to this method, it is not easy to reproducibly prepare model animals or model cells, and it is not always easy to accurately diagnose whether or not the improvement of the medical condition has occurred. The diagnosis requires a predetermined time. Therefore, the screening by the conventional method was inefficient.

[0004]

An object of the present invention is to provide a novel cell useful for efficiently screening an agent useful as an anticancer agent or an agent for suppressing cancer metastasis.

[0005]

In recent years, the inventors of the present invention have
We paid attention to the fact that a relationship between sulfated Lewis antigen and malignant tumor was suggested. For example, in human colon cancer tissue, 3'-sulfo Lewis a and / or 3'-sulfo Lewis x antigen epitopes are detected in the cells and in the surrounding non-malignant epithelium (Izawa, M. et al. , (2000) Cancer R
es. 60, 1410-1416), and the 3'-sulfo Lewis x antigen epitope is the major carbohydrate motif in human colon cancer cells (Capon, C. et al., (1997) J. Biol. Chem.
272, 31957-31968) has been reported. In view of these, the inventors of the present invention have deficient in the enzyme required for the sulfation of Lewis x antigen or Lewis a antigen, and thus, have not been sulfated Lewis x
Candidates for cells that have the ability to produce sulfated Lewis x antigen or sulfated Lewis a antigen by introducing a gene encoding the enzyme into a host cell that cannot produce the antigen or sulfated Lewis a antigen By administering a drug and screening a drug that inhibits the production of sulfated Lewis x antigen or sulfated Lewis a antigen, it is possible to screen a drug effective as an anticancer drug or a cancer metastasis inhibitor. In addition, they succeeded in actually producing such cells and completed the present invention.

That is, according to the present invention, by introducing a gene for at least one enzyme required for sulfation into a host cell lacking the enzyme required for sulfation of Lewis x antigen or Lewis a antigen, Provided is a sulfated Lewis x antigen-producing cell or a sulfated Lewis a antigen-producing cell having an ability to produce a modified Lewis x antigen or Lewis a antigen.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION First, the meaning of the abbreviations used in the present specification will be described. CHO cells: Chinese hamster ovary cells MES: 2- (N-morpholino) ethanesulfonic acid PA: 2-aminopyridine PAPS: 5'-phosphoadenosine 3'-phosphosulfate ORF: open reading frame Lc4: lacto-N-tetraose Galβ1-3GlcNAcβ1
-3Galβ1-4Glc nLc4: Galβ1-4GlcNA, a lacto-N-neotetraose
cβ1-3Galβ1-4Glc lacto-N-fucopentaose II: Galβ1-3 (Fucα1-4) Glc
NAcβ1-3Galβ1-4Glc Lacto-N-fucopentaose III: Galβ1-4 (Fucα1-3) Gl
cNAcβ1-3Galβ1-4Glc Le ^a : Lewis a antigen, ie Galβ1-3 (Fucα1-4) GlcN
Ac-R Le ^x : Lewis x antigen: i.e. Galβ1-3 (Fucα1-3) GlcN
Ac-R

[0008] As described above, the sulfated Lewis x antigen-producing cells and sulfated Lewis a antigen-producing cells of the present invention are added to a sulfase deficient host cell required for sulfation of Lewis x antigen or Lewis a antigen. By introducing a phosphatase gene, the ability to produce sulfated Lewis x antigen or Lewis a antigen is imparted. Here, the sulfating enzyme required for sulfating the Lewis x antigen or the Lewis a antigen is not particularly limited, but the sulfate is transferred to the 3'position of the β-galactosyl residue of the oligosaccharide containing the non-reducing β-galactoside. Sulfotransferase and α1,4-fucosyltransferase III (hereinafter referred to as “FucT-III”) can be mentioned. It becomes possible to produce x antigen or sulfated Lewis a antigen.In the present specification, the "gene" of an enzyme means a nucleic acid encoding the enzyme and also includes cDNA.

The present applicant has previously described a sulfotransferase (designated as "GP3ST") that transfers sulfate to the 3'position of the β-galactosyl residue of an oligosaccharide containing a non-reducing β-galactoside, and a cDNA encoding the same. Patent application (Japanese Patent Application 2001-105201) (Honke, K. et al., (2001) J. Bi
ol. Chem. 276, 267-274). In the present invention, the GP3ST gene can be preferably used. The nucleotide sequence of GP3ST gene and the amino acid sequence encoded by it are shown in SEQ ID NO: 1 in the sequence listing. On the other hand, the FucT-III cDNA was cloned in 1990 and its nucleotide sequence is widely known (K
ukowska-Latallo, JF et al., (1990) Genes Dev. 4,
1288-1303).

[0010] These enzymes do not necessarily have to be wild type, and if they have the activity of each enzyme,
Those having mutations can also be used. In the present specification, the term “enzyme” is not limited to a wild-type enzyme, but is also used to include a substance having the activity of the enzyme. Generally, in a protein having a physiological activity such as an enzyme, one or more amino acids in its amino acid sequence are substituted or deleted, or one or more amino acids are inserted or added to the amino sequence. However, it is well known that the physiological activity may be maintained. Therefore, the amino acid sequence of the wild-type enzyme has an amino sequence in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added to the amino sequence, and the activity of the enzyme is A protein possessed by the protein (hereinafter referred to as “mutant enzyme” for convenience) can be used. The amino acid sequence of such a mutant enzyme is similar to that of the wild-type enzyme.
0% or more, preferably 90% or more, more preferably 9
It is preferable to have a homology of 5% or more. The homology of amino acid sequences can be easily calculated using well-known computer software such as FASTA, and such software is also available on the Internet. Further, as the mutant enzyme, an amino acid having a wild-type amino acid sequence or one or several amino acids substituted or deleted in the sequence, or one or several amino acids inserted or added to the amino sequence is used. Those having a sequence are particularly preferred.

As the host cell, any cell that cannot sulphate the Lewis x antigen or the Lewis a antigen because it lacks the enzyme required to sulphate the Lewis x antigen or the Lewis a antigen can be used. Yes, mammalian cells are preferably used. In the present invention, examples of cells usable as host cells include CHO cells and human lung cancer cells A.
549, human colon cancer cell COLO201 and the like, but not limited to these.

Introduction of a foreign gene into a host cell can be easily carried out by a well-known method. Various expression vectors for mammalian cells are commercially available. By introducing an enzyme gene into the cloning site of these expression vectors and transfecting mammalian cells with the obtained recombinant vector, The enzyme gene can be easily introduced. In addition, the enzyme gene used in the present invention has a known nucleotide sequence of its cDNA.
Using a pair of primers that hybridize to both ends of the ORF region, they can be easily prepared in a large amount by a conventional method such as RT-PCR, and can be easily incorporated into the above expression vector. The cells containing the template RNA in this case include human colonic mucosal epithelium for both GP3ST and FucT-III.

Administration of a candidate drug to the cells of the invention,
By screening a drug that inhibits the production of sulfated Lewis x antigen or sulfated Lewis a antigen, a drug effective as an anticancer drug or a cancer metastasis inhibitor can be screened. Whether or not the cells produce sulfated Lewis x antigen or sulfated Lewis a antigen is
An antibody against sulfated Lewis x antigen or sulfated Lewis a antigen, preferably a monoclonal antibody, can be reacted with cells and examined by a conventional method such as flow cytometry. An example of a specific method is described in the examples below.

[0014]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

I. Experimental Method (1) Materials PAPS was purchased from Sigma (St. Louis, Mo., USA). Lacto-N-tetraose and lacto-N-neotetraose were purchased from Seikagaku Corporation (Tokyo). L-Fucose and GDP-Fuc were purchased from Nakarai Tesque (Kyoto). Lc4-PA and nLc4-PA are the GlycoTAG reagent kit (manufactured by Takara Shuzo in Shiga Prefecture) and the automatic pyridyl amination device (manufactured by Takara Shuzo).
GlycoTAG) with lacto-N-tetraose and lacto-N
-Synthesized by the pyridyl amination of neotetraose. FucT-III is a DNA fragment encoding the known human FucT-III ORF (Kukowska-Latallo, JF et al.,
(1990) Genes Dev. 4, 1288-1303) expression vector pSecT
agA (Invitrogen, Carlbad, CA, USA
In pSec-FucT-III constructed by incorporating the company) from conditioned media of transfected CHO cells was isolated using the Ni ²⁺ column chromatography.
Human FucT-IV, -VII and -IX were prepared by known methods (Nishihara
S. et al., (1999) FEBS Lett. 462, 289-294). Human FucT-V and -VI were purchased from Calbiochem (San Diego, CA, USA). Arylsulfatase A is prepared by a known method (Fujii, T., et al., (199
2) Purified from human placenta by Biochim. Biophys. Acta 1122, 93-98).

The GP3ST expression plasmid pcXN2-GP3ST is obtained by converting the ORF region of the human GP3ST cDNA (see SEQ ID NO: 1) into a known expression vector pcXN2 (Niwa H. et al. (1991) Gene 108,
193-199) and built it. pcXN2-GP3ST
Cell lysates of CHO cells transfected with E. coli were used as a source of GP3ST. FucT-III expression plasmid pcDNA-FucT-III
Is the ORF region of human FucT-III (Kukowska-Latallo, JF,
(See above) is a commercially available expression vector pcDNA3.1 / Zeo (+) (Invitroge
(manufactured by company n).

Mouse anti-Le ^a monoclonal antibody MAB2108
(Chemicon, Inc. of the United States Temecula, CA), mouse anti-sialyl-Le ^a monoclonal antibody ZY-CO9 (made by the United States of South San Francisco, Calif Zymed, Inc.), (manufactured by Calbiochem Inc.) mouse anti-Le ^x monoclonal antibody P12, mouse anti-sialyl Le ^x Monoclonal antibody KM93 (Calbioche
m), mouse anti-3'-sulfoLe ^a and 3'-sulfoLe ^x monoclonal antibody SU59 (Mitsuoka, C. et al., (1998) J. Biol.
Chem. 273, 11225-11233) and mouse anti-3'-sulfoLe ^a
Monoclonal antibody 91.9H (Tsuiji, H. et al., (1998)
Biochem. Biophys. Res. Commun. 253, 374-381; Lovele
ss, RW et al., (1998) Glycobiology 8, 1237-1242)
Was used to detect carbohydrate epitopes. Mouse IgG1 (Dako, Carpinteria, Calif., USA) was used as a negative control.

(2) 3'-sulfation of Lc4-PA and nLc4-PA and
3'-sialylated 3'-sulfo-Lc4-PA and 3'-sulfonLc4-PA are recombinant GP3ST
Was used to synthesize Lc4-PA and nLc4-PA by sulfation, respectively, and purified by anion exchange chromatography and reverse phase HPLC chromatography (Honke, K. e.
t al., supra). These substrates have NMR spectra and ES
It was measured by mass spectrometry using a quadrupole ion trap mass spectrometer equipped with an I source (LCQ ion trap mass spectrometer (trade name), manufactured by Thermo Finnegan, San Diego, Calif., USA). Mass spectra were obtained by negative ion detection, 3'-sulfo Lc4-PA and 3'-sulfo nLc4-PA.
Were identified at m / z 864.2 and 864.4, respectively.

3'-Sialyl Lc4-PA and 3'-Sialyl nLc4-P
A was synthesized by α2,3-sialyltransferase from Lc4-PA and nLc4-PA, respectively. The reaction is 37
The reaction is carried out at 80 ° C for 8 hours.
4), 0.2% Triton X-100 (trade name), 0.5 mg / ml BSA, 2.5
mM CMP-NeuAc (Calbiochem), 1 mM Lc4-PA or nLc4-
PA and 0.6 μg of α2,3-sialyltransferase (Ca
(manufactured by lbiochem) in a final volume of 50 μl, and the reaction product was isolated by HPLC (Honke, K. et al., supra). 3'-Sialyl nLc4-PA was identified at m / z 1075.3 by negative detection with a quadrupole ion trap mass spectrometer.

(3) PA-Lc4, PA-nLc4, 3'-sulfo Lc4-P
A, 3'-sulfonLc4-PA, 3'-sialyl-Lc4-PA and 3'-sialyl-nLc4-PA α1,3 / 1,4-fucosylated standard incubation mixture contained the following components in a total volume of 10 μl: Included. 50 mM MES buffer (pH 6.5), 25 mM
MnCl ₂ , 5 mM ATP, 10 mM L-fucose, 75 μM GDP-Fuc,
25 μM of each acceptor substrate (however, 3'-sialyl Lc4
-PA was used at a concentration of 12.5 μM) and 5 μl of purified FucT-II
I. After incubating at 37 ° C. for 2 hours, the reaction was stopped by boiling for 4 minutes. After adding 90 μl of water, the sample was centrifuged at 15000 rpm for 5 minutes and the Shimadzu LC-VP
TSK gel ODS-80 equipped with HPLC system (Shimadzu)
20 μl of the supernatant was injected into a TM column (4.6 x 250 mm, manufactured by Tosoh Corporation), and a fluorescence spectrophotometer (excitation 320 nm, emission 400 nm)
20 mM ammonium acetate buffer (pH 4.0) while monitoring with
Elution at 35 ° C with a flow rate of 0.8 ml / min. Fractions containing 3'-sulfolacto-N-fucopentaose II-PA and 3'-sulfolacto-N-fucopentaose III-PA are pooled, dried,
It was analyzed by mass spectrometry as described above.

Α1,3-fucosyltransferases (FucT-IV, -V, -VI, VII and IX) for type 2 oligosaccharides, nLc4-PA, 3'-sulfonLc4-PA and 3'-sialyl nLc4-PA The enzyme reaction was performed in 20 μl of the following mixture to determine 50 mM MES buffer (pH 6.5), 25 mM MnCl ₂ , 5 mM
ATP, 10 mM L-fucose, 75 μM GDP-fucose, 25 μ each
M acceptor substrate and each enzyme source: FucT-IV, VII and I
X, 6 μl of cell lysate (Nishihara S. et al., Supra);
FucT-V, 1.8 μg (6 μl); 240 ng (0.6 μl) for FucT-VI, nLc4-PA and 3'-sialyl nLc4-PA, 3'-sulfonLc4-PA
Against 48 ng (0.12 μl). The other reaction conditions were the same as above.

(4) CHO cells stably transfected with GP3ST and FucT-III gene or not and A54
Flow cytometric analysis of 9 cells Linearized CHO cells and A549 cells using Effectene (trade name) transfection reagent (Qiagen, Hilden, Germany) according to a standard protocol for stable transfection pcXN2-GP3ST And / or pcDNA-FucT-
III, transfected with G418 (Sigma) and / or
Based on resistance to Zeocin (manufactured by Invitrogen), clones stably expressing these genes were selected, and the enzyme activity was measured by the above method. 10% fetal bovine serum, 100 units / ml penicillin, 0.1 mg /
ml streptomycin, 400 μg / ml G418 and / or 150
Cultured in DMEM medium containing μg / ml Zeocin and containing 1 mM EDTA
It was collected with PBS. 50 μl of cell suspension (5-10 x 10 ⁶ ) was added to primary antibody (1: 5 diluted SU59; 1:25 diluted P12 and KM93; 1:50 diluted M
AB2108, ZY-CO9, 91.9H and control immunoglobulin) and incubated for 30 minutes under ice cooling. Next, the cells are treated with 1 mM P
The cells were washed with BS, resuspended in 100 μl of 1:25 diluted FITC-binding F (ab ′) ₂ fragment of goat anti-mouse immunoglobulin (DAKO), and incubated under ice-cooling for 30 minutes. Flow cytometric analysis was performed on a FACScan instrument (Becton Dickinson, Franlin Lakes, NJ, USA) operated with CELLQuest ™ software.

(5) Western blotting of CHO cells transfected with or without GP3ST or FucT-III gene 1% Triton X-100 (trade name), 1 mM EDTA and 0.1% mammalian cells and tissue extraction Volume of 10 mM Tris-HCl containing protease inhibitor mixture (made by Wako Pure Chemical Industries)
Parental cells and GP3ST and / or FucT in buffer (pH 7.4)
CHO cells transfected with the -III gene were suspended. After incubating under ice cooling for 1 hour, the solution was added to 1500
After centrifugation at 0 rpm for 30 minutes, the supernatant was used as a cell lysate. Protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL). Cell lysates were separated by SDA-PAGE on a 7.5% gel and a nitrocellulose transfer membrane (Schl, Keene, NH, USA) was used.
Eicher & Schuell) and stained with 1: 5 diluted monoclonal antibody SU59 and 1:20 diluted monoclonal antibody KM93. To investigate the sensitivity to N-glycanase,
Prior to incubation with SU59, blot membranes blocked with 3% BSA were treated with 30 units of N-glycanase F (Roche, Versal, Switzerland) for 24 hours at 37 ° C in 4 ml PBS.

(6) 3'-sulfo Lc4-PA, 3'-sulfolacto-
The desulfation reaction mixture of N-fucopentaose II-PA, 3'-sulfonLc4-PA and 3'-sulfolacto-N-fucopentaose III-PA had a total volume of 50 μl, and 0.5 M acetic acid-NaOH buffer (pH 5.0). , 0.6 μM each, in the presence or absence of arylsulfatase A. 37 ° C
After incubating for 2 hours, the reaction was stopped by boiling for 3 minutes. The sample is then centrifuged at 15000 rpm for 5 minutes, 20μ
l of the supernatant was analyzed by HPLC as described above.

Double-transfected CHO cells stably expressing the GP3ST and FucT-III genes were treated with arylsulfatase A. These cells were treated with P containing 1 mM EDTA.
Collect with BS, 150 μl of cell suspension (1-5 x 10 ⁷ cells),
Incubation was performed in the presence or absence of 20 mM acetic acid-NaOH buffer (pH 5.0), 150 mM NaCl and arylsulfatase A. After rotation culture for 12 hours at 37 ℃, 1 ml of cells
The cells were washed 3 times with PBS, resuspended in 50 μl of 1:59 diluted primary antibody SU59, and incubated for 30 minutes under ice cooling. Then, the cells were washed and diluted with F (ab ') ₂ fragment of 1:25 diluted FITC-conjugated goat anti-mouse immunoglobulin under ice cooling for 3
Incubated for 0 minutes. Flow cytometric analysis was performed as described above.

II. Results (1) In Vitro Enzymatic Synthesis of 3'-SulfoLe ^a and 3'-Sulfo Le ^x Structures As described above, GP3ST was isolated from lacto-N-tetraose and lacto-N-neotetraose. Acts on both, but lacto-
N-fucopentaose II and lacto-N-fucopentaose I
It has no effect on any of II. This means that 3'-sulfo-Le
^{In the} biosynthetic pathway of ^a and -Le ^x structure, 3'-of the terminal Gal
This suggests that sulfation precedes α3 / 4 fucosylation of the penultimate GlcNAc. 3'-sulfated G
The α3 / 4-fucosylation of alβ1-3 / 4GlcNAc-R oligosaccharides has only been reported in fragments using a crude enzyme source (Chandra
sekaran, EV et al., (1992) J. Biol. Chem. 267, 2
3806-23814; Degroote, S. et al., (1999) Glycobiolo
gy 9, 1199-1211). Therefore, the present inventors
The biosynthesis of 3'-sulfo-Le ^a and 3'-sulfo Le ^x structures was studied intensively using 3ST and α1,3 / 1,4-fucosyltransferase. Also in the synthetic pathway of 3'-sialyl-Le ^a and -Le ^x , 3'-sialylation occurs before α3 / 4-fucosylation (Hansson, GC and Zopf, D., (1985) J. B.
iol. Chem. 260,9388-9392; Holmes, EH et al., (19
86) J. Biol. Chem. 261, 3737-3743), α-1,3 / 1,4-fucosyltransferase for unsubstituted-, 3′-sialylated and 3′-sulfated Galβ1-3 / 4GlcNAc-R oligosaccharides Compared the effects of.

First, the 3'-sulfated type 1 chain (Galβ1-3GlcNAc-
R) was investigated for α1,4-fucosylation. 3'-sulfo Lc4-P
A was synthesized from Lc4-PA by the catalytic action of GP3ST (Hon
ke, K. et al., supra). Then, 3'-sulfo-Lc4-PA was added to the unique α1,4-fucosyltransferase (Kukowska-Lata
llo, JF et al., supra) and subjected to fucosylation with recombinant FucT-III. In the presence of GDP-Fuc, a prominent product peak appeared at the position indicated by the arrow in Fig. 1b (Fig. 1).
b), but no peak was detected in the absence of the donor substrate (FIG. 1a). The m / z value of the substance in the product peak is 1010.4
And the m of 3'-sulfolacto-N-fucopentaose II-PA
It corresponded to the / z value (c in Figure 1). These results are FucT-
It is shown that III can act on the 3'-sulfated type 1 chain and synthesize ^a 3'-sulfoLea structure. Unsubstituted, 3'-
FucT-II for Sialylated and 3'-Sulfated Acceptors
The efficiency of I was compared (Table 1). This result shows that FucT-III
3'-sulfo Lc4- than unsubstituted Lc4-PA or 3'-sialyl Lc4-PA
Shows preference for PA.

Up to now, six kinds of α1,3-fucosyltransferase isozymes, namely FucT-III, -I
V, -V, -VI, -VII and -IX are known (Nishihara S.
et al., supra); Kudo, T. et al., (1998) J. Biol. Che
m. 273, 26729-26738; Kaneko M. et al., (1999) FEBS.
Lett. 452, 237-242). Since the substrate specificity of α1,3-fucosyltransferase for the 3'-sulfated acceptor has not been investigated, it was investigated in comparison with the specificity for unsubstituted and 3'-sialylated acceptors. When 3'-sulfonLc4-PA was incubated with FucT-III in the presence of GDP-Fuc, 3'-sulfonLc4-PA was synthesized (Fig. 2, e and f). As shown in Table 1, FucT-III is nLc4-PA or 3'-
He preferred 3'-sulfated nLc4-PA over sialyl nLc4-PA, and preferred type 1 chain over type 2 chain as previously reported (Kukowska-Latallo, JF et al., Supra). Then the other α1,3-
The α3-fucosylation of 3′-sulfonLc4-PA by fucosyltransferase was examined. Since the source and specific activity of the fucosyltransferase used are different,
NLc4-PA (FucT-II
I, -IV, -V, -VI and -IX) or 3'-sialyl nLc4-PA (FucT
It was expressed as a relative value to (VII) (Table 2). The preference for sulfated acceptors between α1,3-fucosyltransferases was significantly different from that for sialylated or unsubstituted acceptors. Unlike the case of sialylated acceptors, all α1,3-fucosyltransferases acted on sulfated acceptors. However, the relative reaction efficiency was different depending on the enzyme. FucT-III, -V and-
For VI, 3'-sulfated oligosaccharides were found to be better substrates than unsubstituted or 3'-sialylated oligosaccharides.

The above-mentioned FIGS. 1 and 2 will be described. Lc4-PA (a, b in FIG. 1) or nLc4-PA (d, e in FIG. 2) is
It was incubated with recombinant human FucT-III in the absence (FIG. 1a and FIG. 2d) or presence (DP in FIG. 1b and FIG. 2e) of DP-fucose. The reaction products were isolated by reverse phase HPLC and their fluorescence was monitored as described above. The arrow indicates the elution position of the product. Fractions containing product were pooled, dried and measured by mass spectrometry. Measured m / z of product
Is 3'-sulfolacto-N-fucopentose II-PA (FIG. 1c).
And 3′-sulfolacto-N-fucopentose III-PA (see FIG.
It was consistent with m / z in f).

Table 1 Substrate specificity of α1,3 / 1,4-fucosyltransferase (FucT-III)

[Table 1] Except for ^a 3'-sialyl Lc4-PA, the concentration of acceptor substrate is 25 μM and for 3'-sialyl Lc 4-PA 12.
It was 5 μM. ^b 5 μl of recombinant FucT-III was used for each measurement.

Table 2 Substrate properties of α1,3-fucosyltransferase for unsubstituted, 3'-sialylated and 3'-sulfated acceptors.

[Table 2] ^a Concentration of acceptor substrate is 25 μM. ^b Values are expressed as percentage compared to activity against PA-nLc4. However, for FucT-VII, 3'-sialyl nLc4-PA
It is shown as a percentage compared with the activity against. FucT-III, -IV,
The activities of -V, -VI and IX against nLc4-PA are, respectively,
It was 3.6, 64.9, 6.0 and 144.9 pmol / min / ml. FucT-V
The α1,3-fucosyltransferase activity of II against 3′-sialyl nLc4-PA was 593.0 pmol / min / ml.

(2) Reconstruction of 3'-sulfo Le ^x and Le ^a epitopes in living cells In order to analyze the biological role of 3'-sulfated Lewis epitopes, these epitopes on living cells were analyzed. Expression is required. Therefore, CHO cells were transfected with GP3ST gene and FucT-III gene incorporated into an expression vector, and expression of 3'-sulfated Lewis epitope was detected by flow cytometric analysis using a specific antibody against 3'-sulfated Lewis epitope. Examined. Monoclonal antibody SU59
Recognizes both 3'-sulfo Le ^a and 3'-sulfo Le ^x epitope (Mitsuoka, C. et al., Supra), monoclonal antibodies 91.9H recognizes only 3'-sulfo -Le ^a epitope (Tsu
iji, H. et al., supra; Loveless, RW et al., supra).

Parental CHO cells (FIG. 3, panels a, e and i) and CHO cells transfected with GP3ST gene alone (FIG. 3, panels b, f and j) were recognized by Le ^x (monoclonal antibody P12). 3'-sialyl Le ^x (recognized by the monoclonal antibody KM93) and 3'-sulfo Le
None of the ^x (recognized by monoclonal antibody SU59) was expressed. CHO cells transfected with the FucT-III gene alone expressed Le ^x and 3′-sialyl Le ^x (FIG. 3, panels c and g) but not 3′-sulfo Le ^x (FIG. Panel 3 of 3). This means that CHO cells have βG
It shows that it does not express al 3-O-sulfotransferase. Furthermore, FucT-III-transfected CHO cells showed Le ^a and 3'-sialyl Le ^a (data not shown).
Did not express any of the above. This means that CHO cells express only type 2 chains (Salvini, R. et a.
l., (2001) J. Biol. Chem. 276, 3564-3573).

CHO cells transfected with both the GP3ST gene and the FucT-III gene were SU59 positive (FIG. 3, panel 1) but 91.9H negative (data not shown), which indicates that It is shown that cells express 3'-sulfoLe ^x determinants but not 3'-sulfoLe ^a . This is consistent with CHO cells expressing only type 2 chains. Furthermore, expression of the 3'-sialyl Le ^x epitope on cells transfected with both genes was shown to be 3'- on cells transfected with the FucT-III gene alone.
It was significantly suppressed over the expression of the sialyl Le ^x epitope (Fig. 3, panels g and h). This finding indicates that expression of GP3ST interferes with the biosynthesis of the 3'-sialyl Le ^x epitope in vivo. In FIG. 3, the dotted line shows the result of a negative control using mouse IgG1 as the antibody.

To analyze on which molecule the 3'-sulfo Le ^x epitope is carried, glycoproteins were extracted from CHO cells transfected with GP3ST gene and FucT-III gene, and Western blot analysis was performed. Investigated by. As a result, several protein bands having relatively large molecular weights were specifically stained with the monoclonal antibody SU59. This indicates that the 3'-sulfo-Le ^x epitope was carried on several different proteins. These SU59-positive bands represent anti-3'-sialyl Le in CHO cells transfected with the FucT-III gene alone.
^The band was almost the same as that stained with the antibody KM93. Furthermore, reactivity with anti-3'-sialyl Le ^x antibody was transfected with both GP3ST and FucT-III genes.
It was suppressed in CHO cells. This is consistent with the results of flow cytometry. These findings suggest that 3'-sulfation and 3'-sialylation occur on a common glycoprotein. Furthermore, most of the SU59-positive bands disappeared after N-glycanase treatment. When glycolipids were extracted from double-transfected CHO cells and analyzed by thin layer chromatography-immunostaining, no SU59-positive band was detected (data not shown). These findings, 3'-sulfo Le ^x epitope have shown that it is carried on mainly N- linked sugar on a protein in CHO cells.

[0036] GP3ST the 3'-sulfo Le ^a epitope to examine whether it has the ability to synthesize in living cells, they were transfected into human lung cancer cell line A549. A549 cells
Express the Le ^a antigen, but not with monoclonal antibody 91.9H (a in Fig. 4). When the GP3ST gene was introduced, the cells became 91.9H-positive (FIG. 4b). This is GP3ST
Have shown that they can also synthesize 3′-sulfoLe ^a epitopes in vivo.

[0037] In FIG. 4, as a result of using anti-Le ^a monoclonal antibody MAB2108 is a solid line, the results of using anti-sulfo Le ^a monoclonal antibody 91.9H are shown in bold. The results using mouse IgG1 performed as a negative control are shown by the dotted line.

[0038] (3) 3'-sulfo Le ^x structure on hydrolysis arylsulfatase A sulfate ester bond catalyzes the desulfation of 3-O-sulfo-galactosylceramide containing glycolipids (von Figura, K. et al. ,
(2001) in The Metabolic and Molecular Bases of Inh
erited Disease, eighth edition (Scriver, CR et a
l. eds.), pp.3695-3724, McGraw Hill, New York), so whether this sulfatase is capable of desulfating 3-O-sulfogalactosyl residues on the 3'-sulfo Lewis epitope. Is interesting. As shown by the arrows in c and d of FIG. 6, when 3′-sulfonLc4-PA and 3′-sulfolacto N-fucopentaose III-PA were treated with arylsulfatase A, a peak of desulfation product was detected. Was done. On the other hand, 3'-sulfo Lc4-PA and 3'-sulfolacto-N-fucopentaose II-PA were not desulfated (Fig. 5 a and
b). The results indicate that arylsulfatase A acts on the type 2 chain but not on the type 1 chain. In FIGS. 5 and 6, “ASA” indicates arylsulfatase A, (−) indicates the absence thereof, and (+) indicates the presence thereof. The arrow indicates the position of the desulfated product.

Furthermore, in double-transfected CHO cells
The intensity of the 3'-sulfo Le ^x epitope was determined by arylsulfatase treatment on flow cytometry (Fig. 7).
A slight but significant decrease.

These findings show that the second GlcNAc from the end is
It is shown that arylsulfatase A desulfates 3-O-sulfogalactosyl residues on sulfate-3Galβ1-4GlcNAc-R oligosaccharides, whether or not the residues are α3-fucosylated.

[0041]

INDUSTRIAL APPLICABILITY According to the present invention, sulfated Lewis x antigen-producing cells and sulfated Lewis useful for efficient screening of agents useful as anticancer agents or cancer metastasis inhibitors
a Antigen-producing cells were provided for the first time.

[0042]

[Sequence list]                             SEQUENCE LISTING <110> JAPAN GENOME SOLUTIONS INC. <120> Sulfotransferase for nonreducing β-galactose and nucleic acid en coding the same <130> 01741 <160> 1

[0043] <210> 1 <211> 1613 <212> DNA <213> Homo sapiens <400> 1 ctcacctgcc ccacagccgc accctgcctg tgcctgcacc ctggggagcc cagagccggc 60 aggggccgag gcggtgggac ctcgggggag ctcaagcctc gactgtcccc tcgctggagg 120 ccagaggcca ag atg atg tcc ttg ctg ggc ggc ttg cag aga tac ttc cgg 171               Met Met Ser Leu Leu Gly Gly Leu Gln Arg Tyr Phe Arg               1 5 10 gtc atc ctc ctc ctc ctc ctg gcc ctg act ctg ctc ctg ctg gcc gga 219 Val Ile Leu Leu Leu Leu Leu Ala Leu Thr Leu Leu Leu Leu Ala Gly     15 20 25 ttc ctg cac tcg gac tta gag ctg gac aca ccc ctg ttt ggg ggc cag 267 Phe Leu His Ser Asp Leu Glu Leu Asp Thr Pro Leu Phe Gly Gly Gln 30 35 40 45 gct gag ggg ccg ccg gtc acc aac atc atg ttc ctg aag acg cac aag 315 Ala Glu Gly Pro Pro Val Thr Asn Ile Met Phe Leu Lys Thr His Lys                 50 55 60 acg gcc agc agc acg gtg ctc aac atc ctc tac cgc ttc gcc gag acc 363 Thr Ala Ser Ser Thr Val Leu Asn Ile Leu Tyr Arg Phe Ala Glu Thr             65 70 75 cac aac ctg tcc gtg gcg ctg ccc gcc ggc tca cgc gtc cac ctg ggc 411 His Asn Leu Ser Val Ala Leu Pro Ala Gly Ser Arg Val His Leu Gly         80 85 90 tac ccc tgg ctc ttc ctg gcg cgc tac gtg gaa ggc gtg ggg tcg cag 459 Tyr Pro Trp Leu Phe Leu Ala Arg Tyr Val Glu Gly Val Gly Ser Gln     95 100 105 cag cgc ttc aac atc atg tgc aac cac ctg agg ttc aac ctg cct cag 507 Gln Arg Phe Asn Ile Met Cys Asn His Leu Arg Phe Asn Leu Pro Gln 110 115 120 125 gtg cag aaa gtc atg ccc aac gac acc ttc tac ttc tcc atc ctg agg 555 Val Gln Lys Val Met Pro Asn Asp Thr Phe Tyr Phe Ser Ile Leu Arg                 130 135 140 aac ccc gtg ttc cag ctg gag tcc tcc ttc atc tac tac aaa acc tac 603 Asn Pro Val Phe Gln Leu Glu Ser Ser Phe Ile Tyr Tyr Lys Thr Tyr             145 150 155 gcc ccc gcc ttc cgg ggc gcc ccg agc ctg gac gcg ttc ctg gcc tcg 651 Ala Pro Ala Phe Arg Gly Ala Pro Ser Leu Asp Ala Phe Leu Ala Ser         160 165 170 ccg cgg acg ttc tac aac gac agc cgc cac ctc agg aac gtc tac gcc 699 Pro Arg Thr Phe Tyr Asn Asp Ser Arg His Leu Arg Asn Val Tyr Ala     175 180 185 aag aac aac atg tgg ttc gac ttc ggc ttc gac ccc aac gcg cag tgc 747 Lys Asn Asn Met Trp Phe Asp Phe Gly Phe Asp Pro Asn Ala Gln Cys 190 195 200 205 gag gag ggc tac gtg cgc gcg cgc atc gcc gag gtg gag cgg cgc ttc 795 Glu Glu Gly Tyr Val Arg Ala Arg Ile Ala Glu Val Glu Arg Arg Phe                 210 215 220 cgg ctg gtg ctc atc gcc gag cac ctg gac gag tcc ctg gtg ctg ctg 843 Arg Leu Val Leu Ile Ala Glu His Leu Asp Glu Ser Leu Val Leu Leu             225 230 235 cgg cgc cgg ctg cgc tgg gcg ctg gac gac gtg gtg gcc ttc agg ctc 891 Arg Arg Arg Leu Arg Trp Ala Leu Asp Asp Val Val Ala Phe Arg Leu         240 245 250 aac tcc cgc agc gcg cgc tcc gtg gcc cgc ctg tcg ccc gag acc cgg 939 Asn Ser Arg Ser Ala Arg Ser Val Ala Arg Leu Ser Pro Glu Thr Arg     255 260 265 gag cgc gcg cgg agc tgg tgc gcg ctg gac tgg cgc ctg tac gag cat 987 Glu Arg Ala Arg Ser Trp Cys Ala Leu Asp Trp Arg Leu Tyr Glu His 270 275 280 285 ttc aac cgc acc ctc tgg gcg cag ctg cgc gcc gag ctg ggg ccg cgg 1035 Phe Asn Arg Thr Leu Trp Ala Gln Leu Arg Ala Glu Leu Gly Pro Arg                 290 295 300 cgg ctg cgc ggg gag gtg gag cgg ctg cgc gcc cgg agg cgc gaa ctc 1083 Arg Leu Arg Gly Glu Val Glu Arg Leu Arg Ala Arg Arg Arg Glu Leu             305 310 315 gcg agc ctg tgc ctg cag gac ggc ggc gcg ctc aag aac cac acg cag 1131 Ala Ser Leu Cys Leu Gln Asp Gly Gly Ala Leu Lys Asn His Thr Gln         320 325 330 atc aga gac ccg cgc ctg cgc ccc tac cag tcc ggc aag gcc gac atc 1179 Ile Arg Asp Pro Arg Leu Arg Pro Tyr Gln Ser Gly Lys Ala Asp Ile     335 340 345 ctg ggt tac aac ctc cgg ccg ggc ctg gac aac cag acg ctg ggc gtg 1227 Leu Gly Tyr Asn Leu Arg Pro Gly Leu Asp Asn Gln Thr Leu Gly Val 350 355 360 365 tgc cag agg ctt gtg atg cct gag ctc cag tac atg gcc cgc ctg tac 1275 Cys Gln Arg Leu Val Met Pro Glu Leu Gln Tyr Met Ala Arg Leu Tyr                 370 375 380 gcc ctg cag ttc ccg gag aag ccc ctc aag aac atc ccg ttc ctg ggg 1323 Ala Leu Gln Phe Pro Glu Lys Pro Leu Lys Asn Ile Pro Phe Leu Gly             385 390 395 gcg tagaggggcc gggccgggga cgaggcctcc tgcggacacc agctcctctc 1376 Ala tccgccgtca ccggggaggc cgaggatcct tgcagggctt ctggggcgtt gggaaaccca 1436 ggcccgccgg ccacgactct gccctcagct tcacagtcac cgaagaaatg aagttacagg 1496 aggggcacac cgtccttttg tctctgcctt tgaggttggc gggaactgaa actggtacgg 1556 tctttttgaa tagaaatttg gggtctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1613

[Brief description of drawings]

FIG. 1 shows the enzymatically synthesized 3′-sulfoLe ^a of Example.
It is a figure which shows the elution pattern by HPLC.

FIG. 2 shows 3′-sulfo Le ^x enzymatically synthesized in Examples.
It is a figure which shows the elution pattern by HPLC.

FIG. 3 is a diagram showing the results of flow cytometry of various cells produced in the examples of the present invention.

FIG. 4 is a diagram showing the results of flow cytometry of cells obtained by transfecting GP3ST into human lung cancer cell line A549.

FIG. 5: Incubation of 3′-sulfo-Lc4-PA (FIG. 5a) and 3′-sulfolacto-N-fucopentaose II-PA (FIG. 5b) in the presence or absence of arylsulfatase A. 2 shows a chromatogram obtained by applying reverse phase HPLC.

FIG. 6: Incubation of 3′-sulfonLc4-PA (c in FIG. 6) and 3′-sulfolacto-N-fucopentaose III-PA (d in FIG. 6) in the presence or absence of arylsulfatase A 2 shows a chromatogram obtained by applying reverse phase HPLC.

FIG. 7 shows the results of flow cytometry of CHO cells double-transfected with GP3ST gene and FucT-III gene, with or without arylsulfatase treatment.

Claims (5)

[Claims] 1. By introducing a gene of at least one enzyme required for sulfation into a host cell lacking an enzyme required for sulfation of Lewis x antigen or Lewis a antigen,
Sulfated Lewis x antigen-producing cell or sulfated Lewis with ability to produce sulfated Lewis x antigen or Lewis a antigen
a Antigen producing cell. 2. The sulfated Lewis x antigen or Lewis a antigen is 3′-sulfo Lewis x antigen or 3′-sulfo Lewis a antigen, and the enzyme is β of oligosaccharide containing non-reducing β-galactoside. -The cell according to claim 1, which is sulfotransferase or α1,4-fucosyltransferase III which transfers sulfate to the 3'position of the galactosyl residue. 3. Production of sulfated Lewis x antigen by introducing a gene for at least one enzyme required for sulfation into a host cell lacking the enzyme required for sulfation of Lewis x antigen. The cell according to claim 1, which is a sulfated Lewis x antigen-producing cell imparted with a function. 4. The sulfated Lewis x antigen is 3′-
The cell according to claim 3, wherein the cell is a sulfo Lewis x antigen, and the enzyme is a sulfotransferase that transfers sulfate to the 3'position of the β-galactosyl residue of an oligosaccharide containing a non-reducing β-galactoside. 5. The cell according to any one of claims 1 to 4, wherein the host cell is a Chinese hamster ovary cell or a human lung cancer cell A549.