From the
Department of Urology, Tohoku University
Graduate School of Medicine, Sendai 980-8574 and the
¶Division of Biochemistry, Research Institute,
Miyagi Prefectural Cancer Center, Natori, Miyagi 981-1293, Japan
Received for publication, December 27, 2002 , and in revised form, April 23, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monosialosyl globopentaosylceramide
(MSGb5),1 also known
as stage-specific embryonic antigen-4 (SSEA-4)
(6,
7), is expressed maximally in
murine four-cell stage embryo
(8), in testicular germ cell
tumors (9,
10) and in aggressive cases of
human renal cell carcinomas (RCC)
(11,
12). Recent work on MSGb5
showed that monoclonal antibody (mAb) RM1 specific to MSGb5 stimulated
invasive properties of human breast carcinoma cell line MCF-7
(13). On the other hand MSGb5
(SSEA-4) is also important as a cell surface marker for embryonic stem cells
(14,
15). Therefore, cloning of
2,3-sialyltransferase determining synthesis of MSGb5 may contribute to
understanding not only of testicular carcinogenesis and malignant progression
of RCC and breast carcinoma but also of mouse embryogenesis and stem cell
research.
To clone the cDNA of 2,3-sialyltransferase responsible for synthesis
of MSGb5, which is the first ganglioside in the globo-series ganglioside
pathway (Fig. 1), we utilized a
transient expression cloning system in COS-7 cells according to the procedure
by Seed and Aruffo (16). By
means of this system combined with polymerase chain reaction targeted to
sialylmotif, cDNA coding the gene that determines the expression of MSGb5 was
isolated with mAb RM1, from a cDNA library of human RCC cell line ACHN. The
cloned cDNA was found to be identical to ST3Gal II
(17) in sequence. Analyses of
the newly expressed gangliosides and the sialyltransferase activity in
transfected cells indicate that ST3Gal II is a SSEA-4 (MSGb5) synthase. We
also found that the ST3Gal II mRNA level was indeed increased in most RCC cell
lines and renal tumor tissues examined.
|
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells and Monoclonal AntibodiesThe human RCC cell line ACHN was purchased from Dainihonseiyaku Co. (Osaka, Japan). Human RCC cell lines SMKT-R2, -R3, and -R4 were kindly provided by Dr. N. Miyao (Sapporo Medical College) (19, 20), and TOS-1, -2, and -3 and 3LN were established in our laboratory (21). COS-7 and COS-1 cells were obtained from Human Science Research Resources Bank (Osaka, Japan), and CV-1 cells were from Riken Cell Bank (Wako, Japan). Mouse IgM was purchased from Sigma. The mouse IgM anti-SSEA3 mAb (MC631) was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. The mouse IgM anti-MSGb5 mAb RM1 (18) was purified by ionic exchange chromatography (Takara Shuzo, Kyoto, Japan). Goat anti-mouse IgM was purchased from Zymed Laboratories, Inc. (San Francisco, CA).
Construction of cDNA LibraryDouble-stranded cDNAs were synthesized with oligo(dT) primers using poly(A+) RNA prepared from human ACHN cells. After addition of BstXI adapters (Invitrogen), the cDNA was size-fractionated (>0.4 kb) and ligated into expression vector pME18S, which was kindly provided by Dr. Maruyama (Tokyo Medical and Dental College). The library contained 2 x 106 independent clones in Escherichia coli strain DH10B (Invitrogen).
Isolation of a Human 2,3-Sialyltransferase cDNA
CloneTLC immunostaining and fluorescence-activated cell sorter
(FACS) analysis showed that COS-7 cells express Gb5 and the precursor of MSGb5
but not MSGb5 (Fig. 2). Eight
samples of 7 x 106 COS-7 cells were transfected with 30 µg
of plasmid DNA each in HEPES-buffered saline (20 mM HEPES, pH 7.05,
137 mM NaCl, 5 mM KCl, 0.7 mM
Na2HPO4, 6 mM glucose) by electroporation
using a Gene Pulser II (Bio-Rad) at 220 V and 975 microfarad. Three days after
transfection, the exponentially growing cells were harvested, blocked with 5%
fetal calf serum in PBS for 1 h, and incubated with anti-MSGb5 mAb RM1 (10
µg/ml) for 1 h. The cells were washed twice with PBS and incubated in a
Petri dish (Falcon 1007) coated with anti-mouse IgM goat IgG (Zymed
Laboratories Inc.). Plasmid DNA was rescued from adherent cells by Hirt
extraction (22) and used to
transform E. coli DH10B (Invitrogen) by electroporation. Adherent
cells were observed under the inverted microscope to increase from
100200/dish after the first round of transfection and panning and about
5001000 after the second round to
1 x 104 after
the fifth round. After seven rounds of transfection and panning, we tried to
isolate a cDNA clone that directs synthesis of MSGb5 by sibling selection but
failed to identify positive transfectants. We then employed PCR with
degenerate primers to detect a sialylmotif
(23) that might be
concentrated as the round of transfection and panning advanced. The primers
were sense 5'-TGCCGSCGCTGYGTGGTBGTGGGG and the antisense
5'-GGTGGTYYKGSWSCCAACATC (S = C, G; Y = C, T; B = C, G, T; K = G, T; W =
A, T). PCR was carried out with Ampli Taq Gold (Perkin-Elmer) by 40
cycles of 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1min
[PDB]
. The
PCR product was gradually concentrated as the cycles of transfection and
panning advanced. The concentrated PCR product was isolated and cloned to
pBluescript, and the 14 samples were sequenced. All of the samples of PCR
product were identical to the sialylmotif of ST3Gal II in sequence. Then we
isolated a cDNA of 2.3 kilobase pairs (named pME-Gb5ST) identical to ST3Gal II
in sequence using the amplified PCR product as a probe.
|
PCR of the -Galactoside
2,3-Sialyltransferase cDNAsSo far the three
-galactoside
2,3-sialyltransferases including ST3Gal II have been
reported (17,
24,
25). To determine which
sialyltransferase was most enriched during the cycles of transfection and
panning, we examined the level of the three sialyltransferase cDNA (ST3Gal I,
ST3Gal II, and ST3Gal IV) by PCR in the plasmids extracted from the adherent
cells every cycle of transfection and panning. The primers were sense
5'-CCCGAATTCAAGATGGTGACCCTGCGGAAG and antisense
5'-CCCTCTAGATCATCTCCCCTTGAAGATAAG for ST3Gal I, sense
5'-CCCGGATCCGGCCTGCAGCGCCTCAGCAAG and antisense
5'-CCCGGATCCGCCCCGGTGCCCGATAGATGG for ST3Gal II, and sense
5'-ATGTGTCCTGCAGGCTGGAAGCTC and antisense
5'-TCAGAAGGACGTGAGGTTCTTGAT for ST3Gal IV. PCR was carried as described
above for ST3Gal I and ST3Gal IV and by 30 cycles for ST3Gal II.
DNA SequenceThe cDNA was sequenced with a model 4200L-2 DNA autosequencer (LI-COR) using a Thermo Sequenase cycle sequencing kit (Amersham Biosciences) with fluorescence-labeled M13 forward and reverse primers.
Construction of the Sialyltransferase Ligated into pCEP4 VectorTo examine whether the Gb5ST cDNA encodes the enzyme in RCC cell lines, the cDNA was excised from pME-Gb5ST with XhoI and subcloned into pCEP4 vector (Invitrogen), and the sense and the antisense Gb5ST cDNA ligated into pCEP4 were respectively obtained.
Construction of a Soluble Form of the SialyltransferaseA truncated form of Gb5ST, which lacks 55 amino acids from the NH2 terminus of the open reading frame, was prepared by PCR as described previously using a5' primer containing an in-frame BamHI site and a 3' primer located 50 bp downstream of the stop codon with BamHI site (17). The PCR fragment was subcloned into BamHI site of the pSVL vector containing the protein A (pSVL-protA) kindly provided by Dr. J. C. Paulson (Scripps Research Institute) to yield expression plasmid pSVL-protA-Gb5ST.
Expression of a Soluble Form of the Sialyltransferase and the Enzyme Activity AssayThe expression plasmid (1 µg) was transfected into COS-1 cells on a 60-mm culture dish using Effectene transfection reagent (Qiagen). After 48 h, the cell culture medium collected from three dishes was combined (15 ml), and 5 ml of the culture medium was concentrated by ultrafiltration using a Centricon 10 (Amicon). The remaining 10 ml of the culture medium was mixed with 100 µl of equilibrated IgG-Sepharose beads (Amersham Biosciences), followed by gentle shaking for 12 h at 4 °C. The Sepharose beads were collected by centrifugation and washed three times with serum-free Dulbecco's modified Eagle's medium. The beads were then resuspended in 200 µl of serum-free medium and used for sialyltransferase assay. A part (5 ml) of the medium after incubation with the beads was also collected and concentrated as described above. The original culture medium concentrated, the culture medium concentrated after incubation with the beads, and the beads after incubation with the original culture medium were assayed for sialyltransferase activity using glycolipids as acceptor substrate. The reaction mixture contained 10 mM MgCl2, 0.2% Triton X-100, 50 mM sodium cacodylate buffer, pH 6.5, 2 mM CaCl2, 0.2 mM 2,3-dehydro,2-deoxy-N-acetylneuraminic acid (Sigma), 10 µlof1mM CMP-[14C]NeuAc (Amersham Biosciences), 23.5 µl of the concentrated culture medium or 10 µl of the beads plus 13.5 µl of the medium, and 0.1 mM acceptor in total volume of 50 µl. After incubation at 37 °C for 2 h, the reaction was terminated by adding 200 µl of PBS. The product was isolated by C18 Sep-Pak cartridge (Waters, Milford, MA) and subjected to chromatography on a high performance thin layer chromatography plate (Baker) with a solvent system of chloroform, methanol, and water (50:40:10) containing 0.05% CaCl2. The radioactivity on a plate was visualized with a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).
Stable Transfection with the Sense or Antisense Gb5ST cDNA Ligated into pCEP4 The sense or antisense Gb5ST cDNA was stably transfected into ACHN cells to examine whether the sense cDNA further increases MSGb5 level and whether the antisense cDNA down-regulates MSGb5 level. For transfection, Effectene transfection reagent was used as recommended by the supplier. Stable transfectants were selected under hygromycin (300 µg/ml).
RNA Preparation and Northern Blot AnalysisTotal RNA was prepared respectively from RCC cell lines, nontumor kidney tissues, and renal tumor tissues by the acid guanidium-phenol-chloroform method (26). 5 µg of total RNA from cell line or 15 µg of that from surgically obtained tissue was separated on 1.0% agarose-formaldehyde gel and then transferred to Hybond-N+ membrane (Amersham Biosciences). The truncated form of ST3Gal II was used as a probe. The membrane was hybridized with labeled cDNA probe at 65 °C in a solution containing 136 mM Na2HPO4, 158 mM NaH2PO4, 7% SDS, 1 mM EDTA, and 10 µg/ml salmon sperm DNA and then washed in 2x SSC solution containing 0.1% SDS at 65 °C. The radioactivity was visualized with a BAS 2000 image analyzer (Fuji Film). We obtained the informed consent from the patients prior to the experiments.
FACS AnalysisCells untreated or transfected with the plasmids were harvested by 0.02% EDTA in PBS, incubated with primary antibody for 90 min on ice, washed twice with 1% bovine serum albumin in PBS, and then incubated with fluorescein isothiocyanate-conjugated goat F(ab')2 directed to mouse IgG and IgM (Tago, Burlingame, CA) for 45 min on ice. After washing twice, the cells resuspended in PBS were subjected to FACS analysis.
Isolation and Analysis of Gangliosides from CellsPurification of gangliosides and TLC immunostaining were performed as described before (18).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression of MSGb5 Synthase cDNATo investigate whether the
cloned cDNA directs MSGb5 expression, the expression plasmids pME-Gb5ST and
pCEP4-Gb5ST were transiently transfected into COS-7 and ACHN cells,
respectively. Because COS-7 cells were found to have high sialidase activity,
COS-7 cells transiently transfected by pME-Gb5ST were cultured with sialidase
inhibitor, 2,3-dehydro,2-deoxy-N-acetylneuraminic acid (Sigma) (200
µM). The culture medium with the inhibitor was changed every 24
h before FACS analysis. After 48 h the cells were harvested, incubated with
RM1, and subjected to FACS analysis. With sialidase inhibitor, COS-7 cells
transfected with Gb5ST cDNA showed a significant increase of MSGb5 compared
with those transfected with vector (Fig.
4), which was reproducible in four experiments even though the
difference was not remarkable (1014%). On the other hand, the increase
of MSGb5 was only 25% for COS-7 cells without sialidase inhibitor and
7% for ACHN cells compared with those transfected with vector (data not
shown).
|
To confirm that the cDNA clone encodes MSGb5 synthase, we constructed a soluble recombinant form of the enzyme. As shown in Fig. 5 (a and b), the concentrated cell culture medium from COS-1 cells transiently transfected with the plasmid (pSVL-protA-Gb5ST) showed the transfer activity of [14C]NeuAc to Gb5, asialo-GM1, and GM1a, but no radioactivity was detected toward the other glycolipids. To further verify that the sialyltransferase activity toward Gb5 was derived from pSVL-protA-Gb5ST, the culture medium after incubation with IgG-Sepharose beads and the beads after incubation with the original culture medium were used for sialyltransferase assay. Sialyltransferase activity was only detected for the fractions adsorbed to the beads but not in the nonadsorbed factions (the culture medium after incubation with the beads) (Fig. 5c). In this experiment, less than 40% of the total activity in the culture medium was recovered in the adsorbed fractions, the low recovery being probably due to partial inhibition of the reactions by the beads themselves. These data indicate that the secreted proteins encoded by the cDNA directly catalyze MSGb5 formation.
|
The antisense Gb5ST cDNA ligated into pCEP4 was then stably transfected into ACHN cells to confirm whether the antisense cDNA down-regulates the MSGb5 level. FACS analysis showed that expression of MSGb5 was suppressed in the stable transfectants with the antisense cDNA (Fig. 6a). To further confirm that the level of MSGb5 was decreased, TLC analysis was carried out. Total glycolipids corresponding to the same wet weight (10 mg) from the stable transfectants were subjected to chromatography on the TLC plate and stained by orcinol sulfuric acid. MSGb5 level from each transfectant was determined by comparing with standard MSGb5, the concentration of which was calculated in advance. The densitometric analysis were performed using Scion image (Scion Co.), and the values were normalized to Gb5 showing almost the same level in all the transfectants. The assessed amounts of MSGb5 from the stable transfectants were 71 (vector), 48 (AS-Gb5ST-1), and 52 (AS-Gb5ST-2) pmol/total glycolipid from 10 mg of cell pellets, respectively. (Fig. 6b). Thus, TLC confirmed the significant decrease in MSGb5 synthesis by the antisense cDNA. Unexpectedly, precursor (Gb5) accumulation was not observed with the antisense cDNA compared with vector (Fig. 6b). In an attempt to further increase MSGb5 level in ACHN cells, stable transfection with the sense cDNA was performed. However, isolation of stable transfectants with the sense cDNA has not been successful because of reduced viabilities.
|
ST3Gal II Level in RCC Cell Lines and Renal Tumor TissuesTo examine whether the sialyltransferase is involved in renal carcinogenesis, we analyzed the levels of ST3Gal II mRNA along with those of MSGb5 in human RCC cell lines. A high level of ST3Gal II mRNA was detected in all human RCC cell lines except SMKT-R2, although levels of ST3Gal II mRNA were not necessarily consistent with those of MSGb5 (Fig. 7). When the ST3Gal II mRNA level was examined in nontumor kidney and renal tumor tissues, the level was higher in all six cases of RCC than nontumor kidney tissues (Fig. 8).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nevertheless, the gradual increase of cells (COS-7 cells expressing SSEA-4)
adherent to the Petri dish was observed under the microscope during the
expression cloning procedure, and therefore we attempted to isolate a positive
clone by utilizing the adherent cells. For this purpose we used the
information of the sialylmotif
(23) with the expectation that
if a sialyltransferase cDNA directs synthesis of MSGb5, enrichment of this
cDNA should be observed as the round advances. With degenerate primers, we
found that only a sialylmotif identical to that of ST3Gal II was concentrated
but not the other types of -galactoside
2,3-sialyltransferase.
In confirmation of the clone directing MSGb5 synthesis, we transfected the cDNA into COS-7 cells and ACHN cells. Only a slight increase of MSGb5 was observed in COS-7 cells transiently transfected with the ST3Gal II cDNA, but the addition of a sialidase inhibitor resulted in a significant increase (1014%) of MSGb5. Detection of a higher amount of the product with the inhibitor is probably due to protection from degradation. We do not know at present why transfection of the sense cDNA in COS-7 cells led to a smaller amount of MSGb5 synthesis than expected even with a sialidase inhibitor. However, these results indicate that in addition to the specific antibody RM1, the sialylmotif was a powerful tool to identify the clone with a slight increase of MSGb5 expression.
On the other hand, the MSGb5 level was suppressed in the stable transfectants with the antisense Gb5ST cDNA in RCC cell line ACHN. Unexpectedly, anticipated accumulation of Gb5 was not observed in these stable transfectants compared with that with vector. A possible mechanism for this result may be due to physiological importance to maintain Gb5 under a certain level in ACHN cells.
Transfection of the soluble recombinant form of ST3Gal II demonstrated the
sialyltransferase activity toward Gb5, asialo-GM1, and GM1a in the culture
medium. Concentration of the activity with IgG-Sepharose beads indicates that
it is derived from soluble recombinant form of ST3Gal II (pSVL-protA-Gb5ST).
Thus, the results of our study shows that ST3Gal II has a role of catalyzing
transfer of sialic acid to Gal1,3GalNAc epitope of Gb5 in addition to
that of asialo-GM1 and GM1a
(17), which suggests that the
different glycolipid series, i.e. globo- and ganglio-series, could be
sialylated by the same enzyme. Recently
2,3-sialylation of
GalNAc
1,3Gal determinant by ST3Gal II was demonstrated
(28), indicating that ST3Gal
II is a multifunctional enzyme. The fact that levels of ST3Gal II mRNA are not
necessarily consistent with those of MSGb5 in RCC cell lines may also suggest
that the enzyme plays the other roles.
The level of ST3Gal II mRNA was not necessarily consistent with that of MSGb5 in each RCC cell line. This may also mean that not only sialyltransferase but also the other glycosyltransferases involved in synthesis of globo-series glycolipid (29, 30) or sialidases determine the level of MSGb5. Interestingly all but one of the RCC cell lines examined possessed a high level of ST3Gal II mRNA. Moreover, the levels of ST3Gal II mRNA were higher in all six cases of human RCC tissues than nontumor kidney tissues, which possessed little if any ST3Gal II mRNA. Along with the fact that ST3Gal II mRNA level was very low in normal kidney (17), an increased level of this sialyltransferase mRNA may be related to carcinogenesis of the kidney, although further studies on many cases are necessary. However, surgically obtained renal tissues could not exhibit the exact levels of mRNA, because the time from ligation of renal artery to extirpation usually requires 1 to 2 h or more in radical nephrectomy, and thus mRNAs in renal tissues surgically obtained might be degradated. We have to take this possibility into consideration in analyzing the mRNA level in human materials.
We previously demonstrated that the higher gangliosides are related to the metastatic potential of human RCC (11, 12, 18, 27, 31). Globo-series gangliosides including MSGb5 are the major components of these gangliosides. In this regard decreased motility of ACHN cells stably transfected with pCEP4 antisense Gb5ST was observed compared with those stably transfected with pCEP4 only,2 consistent with the findings by Steelant et al. (13). Thus, increased levels of ST3Gal II mRNA may be involved not only in carcinogenesis of kidney but also in the malignant progression of human RCC. This may indicate that at the time of carcinogenesis, as suggested by increased level of ST3Gal II mRNA, renal carcinoma cells may be ready for the next malignant progression. Studies on the levels of ST3Gal II mRNA in many cases of RCC and on the roles of the enzyme will further shed light on the nature of carcinogenesis as well as the malignant potential of RCC. So far there has been only a study on sialyltransferase in human RCC (32). Down-regulation of ST3Gal IV mRNA was associated with the malignant progression of RCC, and the cases with high level of ST3Gal IV mRNA showed a favorable prognosis (32). In this context, the expression level of ST3Gal II mRNA is likely in inverse relation with that of ST3Gal IV mRNA in human RCC. Because regulated expression of sialyltransferases was observed in human normal tissues (24), regulation mechanisms of sialyltransferase expression were assumed to be involved in carcinogenesis. Elucidation of the mechanisms may be essential to understand the nature of the malignant progression.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Dept. of Urology, Tohoku
University Graduate School of Medicine, Seiryo-machi Aobaku, Sendai 980-8574,
Japan. Tel.: 81-22-717-7278; Fax: 81-22-717-7283; E-mail:
ssaito{at}uro.med.tohoku.ac.jp.
1 The abbreviations used are: MSGb5, monosialosyl globopentaosylceramide;
SSEA-4, stage-specific embryonic antigen-4; Gb5, globopentaosylceramide; RCC,
renal cell carcinoma; mAb, monoclonal antibody; FACS, Fluorescence-activated
cell sorter analysis; PBS, phosphate-buffered saline; ST, sialyltransferase;
TLC, thin layer chromatography.
2 H. Aoki, S. Saito, A. Ito, K. Mitsuzuka, M. Satoh, and Y. Arai, unpublished
results.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|