Takashi Saito, Eiji Miyoshi, Ken Sasai,
Norihiko Nakano, Hironobu Eguchi, Koich
Honke and Naoyuki Taniguchi
J. Biol. Chem. 2002, 277:17002-17008.

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GLYCOBIOLOGY AND
EXTRACELLULAR MATRICES:A Secreted Type of β1,6-N-Acetylglucosaminyltransferase V (GnT-V)
Induces Tumor Angiogenesis without
Mediation of Glycosylation: A NOVELFUNCTION OF GnT-V DISTINCT FROMTHE ORIGINALGLYCOSYLTRANSFERASE ACTIVITY

THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 19, Issue of May 10, pp. 17002–17008, 2002
Printed in U.S.A.

A Secreted Type of ␤1,6-N-Acetylglucosaminyltransferase V (GnT-V)
Induces Tumor Angiogenesis without Mediation of Glycosylation
A NOVEL FUNCTION OF GnT-V DISTINCT FROM THE ORIGINAL GLYCOSYLTRANSFERASE ACTIVITY*
Received for publication, January 17, 2002, and in revised form, February 28, 2002
Published, JBC Papers in Press, February 28, 2002, DOI 10.1074/jbc.M200521200

Takashi Saito, Eiji Miyoshi, Ken Sasai, Norihiko Nakano, Hironobu Eguchi, Koich Honke,
and Naoyuki Taniguchi‡
From the Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan

Angiogenesis represents an obligatory step in cancer progression (1, 2). A variety of factors, such as fibroblast growth factor-2(FGF-2),1 vascular endothelial growth factor (VEGF) and interleukin-8, contribute to tumor growth. The production of thesefactors and cytokines is controlled by complicated mechanisms,
which include increased gene expression, posttranslational modifications, and interactions with the extracellular matrix.
Many growth factors and their receptors, some of which playa role in tumor angiogenesis, are glycoproteins. Recent studies
employing glycosyltransferase gene manipulation have revealed that changes in the oligosaccharide structure of thesereceptors bring about alterations in intracellular signaling,
thus leading to cellular transformation (3– 6). ␤1,6-N-acetylglucosaminyltransferase V (GnT-V or mannoside acetylglucosaminyltransferase 5 (Mgat5)), which catalyzes the formation of␤1– 6 branching on N-glycans, has been proposed as one of themost important glycosyltransferases associated with tumor
metastasis (7, 8). Furthermore, a recent study of GnT-V-deficient mice indicates that it is essential for tumor metastasis as
well as for tumor growth (9).
* This work was supported by Grant-in-Aid for Scientific Research (S)No. 13854010 from the Japan Society for the promotion of Science. The
costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
‡ To whom correspondence should be addressed. Tel.: 81-6-68793421; Fax: 81-6-6879-3429; E-mail: proftani@biochem.med.osaka-u.
ac.jp.
1
The abbreviations used are: FGF, fibroblast growth factor; VEGF,
vascular endothelial growth factor; GnT-V, ␤1,6-N-acetylglucosaminyltransferase V; HSPG, heparan sulfate proteoglycan; HUVEC, humanumbilical vein endothelial cell(s); PlGF, placental growth factor; CAM,
chorioallantoic membrane.

Clinical studies have shown increases in GnT-V activity inbreast and hepatocellular carcinomas (10, 11). In human breast
cancer cells, a positive correlation was observed between
GnT-V activity and tumor size (11). We have found that theexpression of GnT-V in human colon cancer tissues was correlated with a poor prognosis and distant metastasis (12). Thissuggests that GnT-V level should be indicative of a poor prognosis in cases of colorectal cancer. These results strongly suggest that GnT-V plays a pivotal role in tumor malignancy.
However, the detailed mechanisms of the regulation via GnT-V
with respect to tumor size or metastasis remain unknown.
To address this issue, we established GnT-V transfectantsand examined the metastatic potentials of these cells. In thecourse of this study, we found that GnT-V transfectants induced dramatic increase in angiogenic activity. The inductionof tumor angiogenesis by GnT-V is thought to be due to 1)
increases in the expression/production of angiogenic factors, 2)changes in their function via the addition of ␤1– 6 branching,and 3) other unknown mechanisms. In the present study, we
have investigated the mechanisms of tumor angiogenesis by
GnT-V, and the findings herein show that a secreted type of
GnT-V itself was able to induce angiogenesis with no detectable
mediation of glycosylation. In addition, we also found that abasic domain in GnT-V caused the direct release of FGF-2 fromheparan sulfate proteoglycan (HSPG) on the cell surface and/or
extracellular matrix. Our findings here strongly suggest that
GnT-V is a bifunctional protein and that a secreted type of
GnT-V protein itself plays a critical role in tumor angiogenesis,
acting as an angiogenic cofactor of FGF-2.
EXPERIMENTAL PROCEDURES

Cell Cultures and Transfection—A human colon carcinoma cell line,
WiDr was cultured in RPMI 1640 containing 10% of fetal bovine serum(Invitrogen) and antibiotics (penicillin and streptomycin). Gene transfection was performed using the CELL FECTIN™ reagent (Invitrogen).
Human umbilical vein endothelial cells (HUVEC) were isolated as
described previously (13), and cultured in MCDB131 medium (Invitrogen) containing 10% of fetal bovine serum, human FGF-2 (10 ng/ml),
and antibiotics.
Transplantation of the Tumor Cells to Nude Mice—5 ⫻ 105 cells ofeach glycosyltransferase transfectant were injected into the back ofathymic mice. After 1 month, the mice were sacrificed. Tumor formation
and angiogenesis were observed macroscopically.
Chick Embryo Chorioallantoic Membrane (CAM)-Angiogenesis Assay—The CAM assay was performed as described previously with slight
modifications (14, 15). CAMs from 8-day-old fertilized white Leghorn
chicken embryos were used in this assay. The cells were seeded onto acollagen sponge at a density of 1 ⫻ 105 cells and then incubated for 4 h.
Collagen sponges were deposited onto 5-mm round silicon rings on the
CAM. In the case of purified GnT-V proteins, each GnT-V mutant
protein was placed on 1% methylcellulose. Each sample was deposited
onto 5-mm round silicon rings on the CAM. The CAMs were incubatedfor 48 h and photographed using a digital camera.

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Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that showthat ␤1,6-N-acetylglucosaminyltransferase V (GnT-V)
functions as an inducer of angiogenesis that has a noveland completely different function from the originalfunction of glycosyltransferase. A secreted type of
GnT-V protein itself promoted angiogenesis in vitro andin vivo at physiological concentrations. The highly basic
domain of GnT-V induced the release of fibroblast
growth factor-2 from heparan sulfate proteoglycan on
the cell surface and/or extracellular matrix, leading toangiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor
metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the
inhibition of tumor angiogenesis.

A Secreted Type of GnT-V Induces Angiogenesis

17003

HUVEC Proliferation Assay—HUVEC were seeded in a 96-well plate
coated with type I collagen (2 ⫻ 103 cells/well). After 24 h, the medium
was replaced with MCDB131 medium containing 0.1% bovine serum
albumin and starved for 24 h. The medium was then replaced with theconditioned medium from glycosyltransferase transfectants or
MCDB131 medium containing human FGF-2 (Dainippon Pharmaceutical Co., Ltd.), GnT-V⌬73, ⌬188, ⌬233, or ⌬436, with or without aneutralizing antibody against FGF-2 (R & D systems). After 24 h, thecells were incubated with [3H]thymidine (1 ␮Ci/ml) for 8 h. Incorporation was evaluated by a Micro96 Harvester (SKATRON) and thenanalyzed with a MicroBeta-Counter (Wallac). The results represent the
average ⫾ S.E. of samples assayed in six wells. All experiments wererepeated at least three times, and essentially the same results wereobtained in each case.
Preparation of Purified Recombinant GnT-V—Two types of GnT-V
proteins, GnT-V⌬73 and ⌬188, both of which are soluble forms, wereprepared in a baculovirus-insect cell system (16). For the construction ofa transfer plasmid for GnT-V⌬233, the plasmid for GnT-V⌬187 wasdigested with EcoRI and EagI. The resulting 1521-bp fragment, which
includes Glu234–Leu741 of hGnT-V and the C terminus polyhistidine
tag, was then ligated into the EcoRI-EagI site of a transfer vector,
pAcGP67-A (PharMingen). For the construction of a transfer plasmid
for GnT-V⌬436, the plasmid for GnT-V⌬187 was digested with EcoRV
and EagI. The resulting 912-bp fragment, which includes Ile437–Leu741
of hGnT-V and the C terminus polyhistidine-tag, was then ligated into
the EcoRV-EagI site of the pAcGP67-A vector. The resulting transfer
plasmids were transfected into Sf21 cells in order to produce a recombinant virus by methods described previously (17). The recombinant
enzymes derived from the infected Sf21 cells were then purified byNi2⫹-chelating affinity chromatography (16).

SDS-PAGE was performed according to Laemmli (18). Each GnT-V
mutant protein (100 ng) was subjected to 10% SDS-PAGE under reducing conditions. The proteins were visualized by silver staining.
Peptide Synthesis—The KRKRKK peptide, corresponding to aminoacids 264 –269 of human GnT-V, and the FSGGPL peptide (amino acids
291–296) were synthesized using a Peptide Synthesizer A432 (Applied
Biosystems). They were purified using reverse-phase high performanceliquid chromatography, and their mass and purity were verified bymatrix-assisted laser desorption ionization time-of-flight mass spectrometry (Voyager-DE™ RP; PerSeptive Biosystems).
FGF-2 Measurement—The concentration of FGF-2 was measured as
described previously (19). HUVEC cells, seeded at 5 ⫻ 104 cells/wellonto collagen-coated 12-well plates, were washed twice with PBS, andthen the medium was then replaced with MCDB131 plus 0.1% BSA (0.5
ml/well), in the presence or absence of each molecule: GnT-V⌬73, GnTV⌬436, the KRKRKK peptide, the FSGGPL peptide, and heparin. Cells
were incubated for 2 h on a rotating plate at 4 °C. The supernatants
were collected and centrifuged at 3000 rpm for 5 min at 4 °C to remove
debris. These samples were assayed using an FGF-2 enzyme-linked
immunosorbent assay system (R & D Systems) according to the manufacturer’s recommendations.
RESULTS

Transplantation of GnT-V Transfectants Induces Hypervascularization in Athymic Mice—Our previous studies demonstrated that the expression level of GnT-V is highly correlatedwith a poor prognosis in colorectal cancer (12). Therefore, weestablished stable transfectants of it using the human coloncancer cell line WiDr, along with control transfectants of ␤1,4-

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FIG. 1. GnT-V transfectants induce angiogenic response in vivo. A, marked angiogenesis induced by GnT-V-transfected WiDr cells
transplanted subcutaneously into athymic mice. The arrowheads indicate blood vessels. B, CAM assay using collagen sponges that contain theindicated transfectants. C, HUVEC proliferation after treatment with culture medium from the indicated glycosyltransferase transfectants. CTR,
normal fresh medium used in the HUVEC culture as a positive control.

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A Secreted Type of GnT-V Induces Angiogenesis

N-acetylglucosaminyltransferase-III and ␣1,6-fucosyltransferase. WiDr cells express the above glycosyltransferases atvery low or negligible levels. When these transfectants weretransplanted to athymic mice, transplanted tumors of GnT-V
transfectants showed a dramatic hypervascularization, compared with the other transplants (Fig. 1A).
GnT-V Transfectants Induced Angiogenesis—To verify the
induction of angiogenesis by the GnT-V transfectants, the chorioallantoic membrane of chick embryo (CAM assay) was employed (14, 15). An increased invasion of blood capillaries into
the collagen sponge was observed only in the case of the GnT-V
transfectants (Fig. 1B). This angiogenesis was also observed
when the GnT-V gene was transiently expressed in WiDr,
COS-1, and Chinese hamster ovary cells (data not shown).
These data suggest that the induction of angiogenesis is acommon effect of GnT-V gene transfection rather than a unique
phenomenon limited to the WiDr clones.
Conditioned Medium from GnT-V Transfectants Stimulates
HUVEC Proliferation—To evaluate the induction of angiogenesis in the GnT-V transfectants, we measured their effects
on DNA synthesis in human umbilical vein epithelial cells(HUVEC) (20). DNA synthesis of HUVEC was increased as theresult of replacement with the conditioned medium from the
GnT-V transfectants, whereas no effects were detectable when
the conditioned medium from the other transfectants was used
(Fig. 1C). These data indicate that the GnT-V transfectants
secreted a growth-stimulating factor for HUVEC. The additionof fresh medium (CTR) increased the HUVEC proliferation to ahigher level than that of the conditioned medium from the
GnT-V transfectants. This is probably due to a supply of
growth-stimulating factors such as FGF-2 that are contained infetal bovine serum.
Effect of Recombinant GnT-V on HUVEC Proliferation—
Next, angiogenic activity in the conditioned medium from the
GnT-V transfectants was characterized using column chromatography, monitoring HUVEC proliferation-stimulating activity. With heparin affinity chromatography, a high activity fraction was eluted with 0.3 M NaCl (data not shown). This
characteristic is completely different from hitherto known angiogenic factors (e.g. FGF-1, FGF-2, VEGF, placental growth
factor (PlGF), and hepatocyte growth factor), which are eluted
with 0.8 –1.5 M NaCl (21–25). When Western blot analysis of
the eluted fractions was performed using an anti-GnT-V antibody, its reactivity corresponded to the HUVEC proliferation
activity (data not shown). It is known that GnT-V, as well asother glycosyltransferases, is secreted from tumor cells (26 –30), although the physiological significance of this remains
unknown. To address the hypothesis that a secreted type of
GnT-V itself induces the proliferation of HUVEC, we prepareda special type of recombinant GnT-V, referred to as GnT-V⌬73,
which lacks the transmembrane domain but in which glycosyltransferase activity is retained (16). HUVEC proliferation wasincreased as a result of the administration of GnT-V⌬73 in adose-dependent manner (Fig. 2A). The utilized concentration
appears to be within the physiological range. The concentrationof GnT-V in conditioned medium from the GnT-V transfectants

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FIG. 2. Purified recombinant GnT-V proteins accelerate HUVEC proliferation. A, HUVEC proliferation promoted by the administrationof GnT-V⌬73. B, constructs of deletion mutants of GnT-V. Black box, the basic region; TM, the transmembrane domain; Stem, stem region of
GnT-V. C, SDS-PAGE of purified GnT-V mutants. D, HUVEC proliferation assay after the addition of 100 ng/ml each purified GnT-V mutant
protein.

A Secreted Type of GnT-V Induces Angiogenesis

17005

was determined to be 140 ng/ml on the basis of the specificactivity of GnT-V⌬73. Furthermore, conditioned medium from
B16-F10 mouse melanoma cells, which have a high endogenous
GnT-V activity, contained ⬃70 ng/ml GnT-V. B16-F10 cells also
showed an angiogenic activity similar to the GnT-V transfectants in the CAM assay (data not shown), suggesting that the
GnT-V secreted from B16-F10 cells can stimulate angiogenesis
in this assay system. In addition, the administration of recombinant ␣1,6-fucosyltransferase indicated the absence of any
HUVEC growth-stimulating activity (data not shown). These
data indicate that a secreted type of GnT-V within the physiological concentration range has growth-stimulating activity
for HUVEC.
Domain Analysis of GnT-V Affecting HUVEC Proliferation—To determine which domain of GnT-V contains the
HUVEC growth-stimulating activity, we analyzed several
types of deletion mutants of GnT-V (Fig. 2, B and C). GnTV⌬73, ⌬188, and ⌬233 mutants stimulated HUVEC proliferation, whereas ⌬436 did not (Fig. 2D). GnT-V⌬73 and ⌬188 have
GlcNAc transferase activity, but ⌬233 and ⌬436 do not. These
data indicate that the HUVEC growth-stimulating activity is
located in the region corresponding to amino acids 234 – 436 of
GnT-V, which does not encompass glycosyltransferase activity.
Identification of a Basic Amino Acid-clustered Region of
GnT-V to Induce Angiogenesis—There is a markedly basic region, corresponding to amino acids 254 –269, of human GnT-V,
whose sequence, KSLAEKQNLEKRKRKK, is very similar tothe sequence of amino acid 142–157 of VEGF189 (21) (Fig. 3A).
In addition, the context of basic amino acids in this region isconserved in PlGF-2 and heparin binding type epidermal
growth factor-like growth factor (HB-EGF) and serves as aheparin-binding motif (21). Barillari et al. (19) reported that abasic peptide, GRGKRR, derived from the sequence of PlGF-2,
induced the growth of endothelial cells by releasing FGF-2 from
HSPG on the cell surface and/or extracellular matrix. Therefore, we synthesized a basic peptide, KRKRKK, corresponding
to amino acids 264 –269 of GnT-V and a nonbasic control peptide, FSGGPL (corresponding to amino acids 291–296 of
GnT-V), and examined their effects on the growth of HUVEC.
The amount of FGF-2 released from HSPG on HUVEC wasmeasured after various truncated GnT-Vs and synthesizedpeptides were administrated to a culture medium of HUVEC
at 4 °C. GnT-V⌬73 and peptide KRKRKK induced the releaseof FGF-2, whereas GnT-V⌬436 and peptide FSGGPL had noeffect (Fig. 3B). Both GnT-V⌬188 and ⌬233, as well as
GnTV⌬73, also induced the release of FGF-2 (data notshown). Similarly, heparin, which is known to release HSPGbinding molecules by competing for their heparin-bindingsite (31), also induced the release of FGF-2. The phosphorylation of FGF receptors on HUVEC by stimulation of thereleased FGF-2 was confirmed (data not shown). The peptide
KRKRKK promoted the growth of HUVEC to an extent similar to GnT-V⌬73 (Fig. 3C). This effect was completely suppressed by the co-addition of a neutralizing antibody against
FGF-2 (Fig. 3C). These results suggest that the KRKRKK
region is sufficient for HUVEC growth-stimulating activity
and that the GnT-V protein stimulates angiogenesis by re-

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FIG. 3. The basic region of GnT-V induces angiogenesis. A, alignment of the basic region of GnT-V with the heparin-binding domains of
VEGF189, PlGF-2, and HB-EGF. The initial amino acid in each protein is numbered at the left. Gray boxes, basic amino acids lysine (K) and arginine(R). Boxes, basic amino acids that are identical in at least three proteins. B, the amount of FGF-2 released from HUVEC after treatment with
GnT-V⌬73 (100 ng/ml), ⌬436 (100 ng/ml), KRKRKK peptide (1 ng/ml), FSGGPL peptide (1 ng/ml), and heparin (30 ␮g/ml). C, the basic peptide
KRKRKK stimulates HUVEC proliferation. HUVEC cells were stimulated with FGF-2 (5 ng/ml), GnT-V⌬73 (100 ng/ml), GnT-V⌬436 (100 ng/ml),
KRKRKK peptide (1 ng/ml), and FSGGPL peptide (1 ng/ml) in the presence (⫹) or absence (⫺) of a neutralizing antibody against FGF-2 (100 ng/ml).

17006

A Secreted Type of GnT-V Induces Angiogenesis

leasing FGF-2 from HSPG on endothelial cells via the actionof the basic region of the protein.
In Vivo Angiogenesis by GnT-V Protein—The induction ofangiogenesis was also observed in other in vitro angiogenic
assays, such as the capillary-like tube formation (32) and themigration assays (33) using HUVEC (data not shown). In order
to investigate the angiogenic activity of GnT-V ex vivo, a CAM
assay using GnT-V⌬73 protein was performed. GnT-V⌬73 induced angiogenesis of chick microvessels as well as FGF-2 (Fig.
4). Moreover, the KRKRKK peptide even induced a similarangiogenesis, and the induction of angiogenesis by GnT-V⌬73
and peptide KRKRKK was inhibited by treatment with a neutralizing antibody against FGF-2. In contrast, neither
GnTV⌬436 nor the control peptide had any angiogenic activity.
These results indicate that a secreted type of GnT-V and
GnT-V-derived peptide KRKRKK induce angiogenesis via theaction of FGF-2. Considering the results relative to HUVEC
proliferation, the basic region of GnT-V may cause the releaseof FGF-2 from HSPG on endothelial cells.
DISCUSSION

Angiogenesis is one of the key regulatory steps necessary fortumor malignancy. Several endogenous stimulators and inhibitors of angiogenesis have been identified, and the net balanceof these regulators represents the angiogenic phenotype oftumor cells. These include several types of molecules, the functions of which were originally thought to be related to events
other than angiogenesis. For instance, angiostatin and endostatin are produced by the proteolysis from plasminogen andtype XVIII collagen, respectively (34, 35). In the present study,
we found that a secreted type of GnT-V protein induces angiogenesis that is unrelated to the usual glycosyltransferase activity of GnT-V. Although a variety of previous studies indicatethat GnT-V is directly linked to tumor metastasis, the mechanistic details of its action at the molecular level remain unknown (6 –9). Dennis’s group reported that oligosaccharide
structures that are modified by GnT-V on an integrin or T cell
receptor affect cell-cell or cell-extracellular matrix interactionsin the processes of tumor metastasis and the immune system(7, 36). The present study proposes a new mechanism of
GnT-V-related tumor metastasis, which is not mediated byglycosylation. Namely, it appears that GnT-V is capable ofacting as a bifunctional protein. GnT-V is a Golgi enzyme but isalso secreted by some cultivated cells (26, 27). The concentration of GnT-V sufficient to induce angiogenesis is within therange of concentration that is actually observed in the conditioned medium of B16-F10 cells. Our hypothesis regarding theaction of GnT-V in angiogenesis is schematically summarized
in Fig. 5. It is thought that GnT-V secreted from cancer cells

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FIG. 4. GnT-V protein induces in vivo angiogenesis. Shown are CAM assays using methylcellulose disks that contain BSA (1 ␮g) (A), FGF-2(50 ng) (B and C), GnT-V⌬73 (1 ␮g) (D and E), GnT-V⌬436 (1 ␮g) (F), FSGGPL (10 ng) (G), or KRKRKK (10 ng) (H and I), in the presence (C, E,
and I) or absence (A, B, D, F, G, and H) of a neutralizing antibody against FGF-2 (nAb) (1 ␮g).

A Secreted Type of GnT-V Induces Angiogenesis

17007

Ets-1 is one of the most important transcriptional factors in
up-regulating GnT-V gene expression (41), Ets-1 may be atarget for suppressing GnT-V expression. The third is to inhibit
the secretion of GnT-V in cancer cells, although the secretionmechanism remains to be solved. The fourth is to mask thebasic domain of GnT-V with some reactive acidic reagents. The
fifth is the enhancement of the degradation of the secreted
GnT-V by proteolysis. First, the issue of whether GnT-V contributes to cancer progression as a glycosyltransferase or anangiogenic factor needs to be determined in individual cancer
cases. A specific inhibitor for the N-acetylglucosaminyltransferase reaction may solve this problem. In conclusion, we reporton a novel mechanism in which a secreted type of GnT-V
protein itself plays a critical role in tumor angiogenesis, acting
as an angiogenic cofactor of FGF-2.

releases a deposited FGF-2 from HSPG in the extracellularmatrix by competition for binding, and subsequently the released FGF-2 binds to the FGF-2 receptor, forming a ternarycomplex with the receptor and associated heparan sulfate, andthis interaction generates a signal leading to stimulation ofangiogenesis. No GnT-V receptor/binding protein on the HUVEC surface was detected by cross-linking analysis using 125Ilabeled GnT-V, suggesting that GnT-V does not directly bind to
HUVECs but acts indirectly to stimulate signal transduction(data not shown). This hypothesis is supported by the reportthat when endothelial cells were precultured with VEGF, theaddition of a protein containing a basic amino acid cluster like
GnT-V induced a release of VEGF from HSPG (19). Furthermore, the growth-stimulating effect of GnT-V was also observed in other cell lines, such as breast carcinoma cell lines
MCF-7 and MDA-MB231 (data not shown), which are known to
respond to FGF-2 (37, 38). These findings suggest that GnT-V
might act as a general growth factor or a differentiation factor
via a mechanism similar to that proposed here.
The angiogenic potential of GnT-V relates to a basic region
that is conserved in the heparin-binding domains of hitherto
known angiogenic growth factors. It is noteworthy in this respect that VEGF189 contains the same basic peptide KRKRKK
as GnT-V (Fig. 3A). VEGF189 is known to exist on the cellsurfaces as a cell-associated form, distinct from the secretableisoforms of VEGF121 and VEGF165. To associate with a cellsurface, the basic region of the C terminus of VEGF189, including the KRKRKK sequence, is required (39). However, there isno evidence that VEGF189 induces the release of FGF-2 from
HSPG. In contrast, a secretable factor, PlGF-2, which contains
the basic region, induces the release of FGF-2 (19). It appears
that a secretable factor that contains a basic amino acid cluster
might effect a release of a FGF-2 associated with HSPG. Although FGF-2 was identified as a target of the secreted type of
GnT-V in this study, other growth factors associated with
HSPG also might be released by GnT-V.
A considerable body of data has accumulated to date on the
involvement of GnT-V in cancer progression (7–12). Therefore,
inhibiting the action of GnT-V is considered a reasonable strategy against cancer progression. Five mechanisms have been
proposed to suppress GnT-V function. First is the inhibition of
the N-acetylglucosaminyltransferase enzyme reaction in cancer cells by the administration of a specific inhibitor. Actually,
the development of such reagents is ongoing (40). The second
involves the suppression of gene expression of GnT-V. Since

Acknowledgments—We thank Dr. Seiji Takashima for technicalsupport and critical discussions. We are grateful for the criticism of
Dr. Yasuhide Miyamoto. Technical supports were provided by
Dr. Katsuhisa Noda, Hideyuki Ihara, Takatoshi Kitada, Yukinao
Shibukawa, Haruhiko Sakiyama, Susumu Nakahara, and Yoshie
Tawara. We also thank Dr. H. F. Deutsch for critically reading themanuscript.
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FIG. 5. Schematic illustration of the induction of tumor angiogenesis by secreted GnT-V. A secreted type of GnT-V that contains
the basic amino acid-clustered domain competes with FGF-2 to bind
HSPG to the cell surface, resulting in the release of FGF-2 and stimulation of its receptor on the target cells.

17008A Secreted Type of GnT-V Induces Angiogenesis

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