THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 37, Issue of September 10, pp. 38838 –38843, 2004
Printed in U.S.A.

Functional Sialylated O-Glycan to Platelet Aggregation on
Aggrus (T1␣/Podoplanin) Molecules Expressed in
Chinese Hamster Ovary Cells*
Received for publication, June 28, 2004
Published, JBC Papers in Press, July 1, 2004, DOI 10.1074/jbc.M407210200

Mika Kaneko‡, Yukinari Kato‡§, Akiko Kunita§, Naoya Fujita§, Takashi Tsuruo§¶,and Motoki Osawa‡储From the ‡Department of Experimental and Forensic Pathology, Yamagata University School of Medicine, 2-2-2,
Iida-nishi, Yamagata 990-9585, Japan, the §Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032 Japan, and the ¶Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research, 1-37-1, Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan

Aggrus, also called T1␣ and podoplanin, is a novel
platelet aggregation-inducing factor that is expressed invarious carcinoma cells. Aggrus/T1␣/podoplanin is
known to be expressed in lung type I alveolar cells or
lymphatic endothelial cells. However, its physiologicalrole has not been clarified. To assess the attribution of
glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a seriesof Chinese hamster ovary (CHO) cell mutants, N-glycandeficient Lec1, CMP-sialic acid transporter-deficientLec2, and UDP-galactose transporter-deficient Lec8. A
new anti-human Aggrus monoclonal antibody, YM-1,
was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells
induced platelet aggregation, but those on Lec2 andLec8 cells did not. Further, the glycans on Aggrus wereanalyzed by lectin blotting. Aggrus expressed in CHO
and Lec1 cells showed Wheat-germ agglutinin, Jacalin,
and Vicia villosa lectin bindings. Lectin blotting results
indicated that sialylated core 1 structures, sialic acid
plus Gal␤1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure
is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.

The concept has been accepted that tumor metastasis isassociated with platelet-aggregation activity possessed by human and other mammalian cancer cells (1, 2). A previous study
has clarified that membranous 44- and 36-kDa sialoglycoproteins in the cancer cells of mice and humans, respectively,
aggregated platelets with no relation to plasma components(3–5). Ectopic expression of these molecules was observed in avariety of cancer cells including colorectal and testicular tumors, in which up-regulated expression was evident (3, 6). The
molecules, designated as Aggrus, belong to a type-I transmembrane sialomucin-like glycoprotein that consisted of an extracellular domain with abundant Ser and Thr residues as potential O-glycosylation sites, a single transmembrane portion, anda short cytoplasmic tail with putative sites for protein kinase C
and cAMP phosphorylation (3, 7).
Homologous molecules to Aggrus have been identified inde* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
储 To whom correspondence should be addressed. Tel.: 81-23-628-5271;
Fax: 81-23-628-5273; E-mail: mosawa@med.id.yamagata-u.ac.jp.

pendently in several mammalians; T1␣ (7, 8), podoplanin (9),PA2.26 (10), gp40 (11), RANDAM-2 (12) are isolated from rat
alveolar type-I cells in lung, rat glomerular epithelial cell
(podocyte), mouse keratinocytes, Madin-Darby canine kidneycells (type I), and glutamatergic neuronal cells in mouse cerebrum, respectively. Podoplanin (gp36) is utilized as a specificmarker for lymphatic endothelium in histopathology (13, 14).
Ramirez and co-workers (15, 16) showed that T1␣ null mice,
generated by a targeted dysfunction of the gene, died at birthfrom lethal respiratory failure accompanied by immature lymphatic vessel formation (15, 16). T1␣/podoplanin conceivably
plays an important role in regulating peripheral lung cell proliferation and lymphatic vascular development.
In the experiment with respect to the 8F11 antibody thatneutralized the platelet aggregating activity, it was evident
that the segment of EDXXVTPG in the extracellular domain,
designated as platelet aggregation-stimulating domain, wascritical for the activity of Aggrus (3). In particular, this motif,
which is highly conserved among species, is tandemly triplicated in gp40 of dog. The occurrence of O-glycosylation to Thr
residues was proved by Edman degenerative microsequencing(17). Furthermore, in the study of targeted mutagenesis tomouse and human Aggrus molecules, we obtained evidence
that glycans on the platelet aggregation-stimulating domainplay an important role for platelet aggregation (3, 5).
The present study examined the association of glycosylation
with the platelet aggregating activity; recombinant Aggrus
molecules were expressed in a set of three defective cell lines
from Pro-5 Chinese hamster ovary (CHO)1 cells. Those mutant
cells have defects in distinct steps of glycosylation. Lec1 CHO
cells lack the glycosyltransferase termed GlcNAc-TI, which
produces incomplete intermediates of N-linked carbohydrates(18 –20). Lec2 cells are CMP-sialic acid transporter-deficient.
In addition, both glycoproteins and glycolipids lack 90% of
sialic acids (SA). Lec8 cells exhibit a remarkable reduction of
the ability to transport UDP-galactose (Gal) into the Golgi
compartment (22, 23). Platelet aggregation-inducing activity
was examined in those stably transfected cells. Moreover, thestructure of active glycans was examined by the lectin-blot
analysis. In addition, a monoclonal antibody to human Aggrus(hAggrus) was obtained by immunizing rat with the synthetic
hAggrus peptide.

1
The abbreviations used are: CHO, Chinese hamster ovary; SA, sialicacid; Gal, galactose; WGA, Wheat-germ agglutinin; s-WGA, succinylated WGA; VVA, Vicia villosa lectin; mAb, monoclonal antibody; h,human; m, mouse.

38838

This paper is available on line at http://www.jbc.org

Aggrus Activity Is Regulated by Glycosylation

38839

FIG. 1. Characterization of YM-1 and platelet aggregation-inducing activity of cell lines. A, immunohistochemical staining with YM-1
on human specimens derived from prostate. The scale bar represents 10 ␮m. B, the cell lysate of lymphatic endothelial cell, NCI-H226,
CHO-hAggrus, and CHO were electrophoresed and immunoblotted with YM-1 and anti-podoplanin mAb. C, flow cytometric analysis of YM-1 andanti-podoplanin reactivity to CHO-hAggrus, lymphatic endothelial cell, and NCI-H226. D, CHO-hAggrus, lymphatic endothelial cell, and
NCI-H226 were incubated with mouse platelet-rich plasma, and platelet aggregation activity was confirmed. Parental CHO served a control. WB,
Western blot.
EXPERIMENTAL PROCEDURES

Materials—Lymphatic endothelial cell was purchased from AngioBio(Del Mar, CA). Lung carcinoma cells, NCI-H226, and NCI-H520 wereobtained from the American Type Culture Collection (ATCC). The antimouse Aggrus rat monoclonal antibody (mAb), 8F11, was preparedpreviously (24). Mouse anti-human podoplanin (gp36) mAb was obtained
from Research Diagnostics Inc. (Flanders, NJ). Anti-FLAG antibody (M2)
was purchased from Sigma-Aldrich. Horseradish peroxidase-conjugatedsecondary antibodies were obtained from DakoCytomation Co., Ltd.
(Glostrup, Denmark). Fluorescein-isothiocyanate-conjugated anti-rat
IgG was obtained from Cappel (Cochranville, PA). Fluorescein-isothiocyanate-conjugated anti-mouse IgG was from MC Biomedicals (Irvine,CA). Horseradish peroxidase-conjugated streptavidin was obtained
from Amersham Biosciences. The following biotinylated lectins werepurchased from Vector Laboratories Inc. (Peterborough, UK), concanavalin A, Dolichos biflorus agglutinin, peanuts agglutinin, Ricinus

38840Aggrus Activity Is Regulated by Glycosylation

FIG. 2. Flow cytometry of transfected Lec1, Lec2, and Lec8 cells and platelet aggregation-inducing activity of transfected cells. A,
the Lec1 (thin line), Lec2 (thick line), and Lec8 (dotted line) cells were transfected with pcDNA-hAGR and pcDNA-mAGR. Cells were incubated with
YM-1 and 8F11, followed by fluorescein isothiocyanate-labeled secondary antibodies. Parental CHO (shaded) was involved as a control. B,
platelet-rich plasma derived from mouse blood was incubated with the transfected Lec cells that expressed hAggrus and mAggrus. C, microscopic
features of the induced platelet aggregation are also indicated. The scale bar represents 100 ␮m.
communis agglutinin I, soybean agglutinin, Wheat-germ agglutinin(WGA), succinylated WGA (s-WGA), Griffonia simplicifolia lectin I and
II, Sophora japonica agglutinin, Datura stramonium lectin, Jacalin,
Lycopersicon esculentum lectin, Solanum tuberosum lectin, Vicia villosa
lectin (VVA), Sambucus nigra lectin, and Maackia amurensis lectin I
and II.
Hybridoma Production—Two Sprague-Dawley rats were immunizedwith the synthetic peptide, CEGGVAMPGAEDDVV, corresponding toamino acids 38 –51 of hAggrus plus an N-terminal cysteine. The spleencells were fused with mouse myeloma P3U1 cells. Then the culture
supernatants of the hybridomas were screened by enzyme-linked immunosorbent assay for the binding activity to the Aggrus peptide. The
established mAb was designated YM-1.
Immunohistochemistry—YM-1 was added to the deparaffinized andrehydrated specimens for 4 h at 23 °C. Biotin-conjugated secondaryanti-rat IgG antibody was added, followed by incubation with peroxidase-conjugated avidin-streptavidin complex (Vectastain ABC Kit, Vector Laboratories Inc.). Color was developed with 3,3-diaminobenzidine
tetrahydrochloride tablet sets (DakoCytomation Co. Ltd.) used for 10
min. The human specimens were derived from prostate cancer (25).
Production of Aggrus in CHO/Lec Cells—Lec1, Lec2, and Lec8 cellsof CHO mutant cell lines were obtained from ATCC (18 –23). Cells
were cultured at 37 °C in 5% CO2 with ␣-minimal essential medium(Sigma-Aldrich) containing 10% fetal calf serum (Moregate Biotech,
Queensland, Australia), supplemented with L-glutamine (2 mM), L-proline (0.04 mg/ml), penicillin (100 units/ml), and streptomycin (100 ␮g/ml). Human and mouse aggrus cDNAs (AB127958, AJ297944), incorporated with FLAG tag sequence, were subcloned into a vector of
pcDNA3 (Invitrogen), which were named as pcDNA-hAGR andpcDNA-mAGR, respectively. Lec1, Lec2, and Lec8 cells were transfected with pcDNA-hAGR and pcDNA-mAGR by a procedure using
LipofectAMINE reagent (Invitrogen) as described previously (3). Stableproduction was obtained using a selective culture of the transfectants ina medium containing Geneticin (G418; Sigma-Aldrich) at a concentration of 1.0 mg/ml for a couple of weeks.
Flow Cytometry—The expression levels of hAggrus and mouse Aggrus (mAggrus) were confirmed comparatively using by flow cytometry,as described previously (26). Briefly, lymphatic endothelial cells, NCIH226 cells, parental CHO cells, and Lec cells, collected by trypsinEDTA treatment, were incubated with antibodies for 1 h at room
temperature, YM-1 culture supernatant, and 8F11 in phosphate-buffered saline at a concentration of 1 ␮g/ml. Then the cells were incubatedAggrus Activity Is Regulated by Glycosylation

38841

with fluorescein isothiocyanate-conjugated antibodies for 30 min. Flowcytometry was performed using FACS Caliber (BD Biosciences).
Platelet Aggregation—Platelet aggregation was examined by the procedures of Toyoshima et al. (5). Briefly, mouse platelet-rich plasma wasprepared from fresh heparinized blood extracted from BALB/c mice. An
aliquot of 200 ␮l of platelet-rich plasma was incubated in a cuvette at
37 °C with continuous stirring. After 5 min, 5 ␮l of phosphate-bufferedsaline-washed cells were added. Then light transmittance was monitored at 660 nm for 10 min. This assay was performed with a NKKHEMA Tracer I (Niko Bioscientific Co., Tokyo, Japan). A photograph
was taken using a phase contrast microscope (100⫻, microphoto Fxa;
Nikon Corp.) at the same time.
Western Blot Analyses and Lectin Blotting—Cultured cell pellets werelysed with a buffer for 30 min on ice. The buffer consisted of 25 mM Tris,
50 mM sodium chloride, 0.2% SDS, 0.5% sodium deoxycholate, 2% Nonidet
P-40, 50 ␮g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH
7.4. The lysate supernatants were centrifuged for 15 min at 15,000 rpm to
remove cellular debris and nuclei. Cell lysates containing 5 ␮g of total
protein were prepared for Western blot analysis by boiling in SDS sample
buffer (50 mM Tris, 2% SDS, 5% glycerol, 10% 2-mercaptoethanol, pH 6.8).
For lectin blotting analysis, cell lysates were immunoprecipitated with
anti-FLAG antibody conjugated agarose for 1 h at 4 °C. After the complexes were washed, elution was performed with a SDS sample buffer byboiling for 5 min, followed by polyacrylamide gel electrophoresis in 10 –20% gradient gels. After proteins were transferred onto an immobilon
polyvinylidene difluoride membrane (Millipore Corp., Billerica, MA), thestrips were incubated with YM-1 antibody or biotinylated lectins (20␮g/ml) for 1 h at room temperature followed by incubation with horseradish peroxidase-conjugated anti-rat IgG or streptavidin coupled to peroxidase. The enzyme activity was developed using an enhanced chemiluminescence kit (ECL, Amersham Biosciences).
RESULTS

Establishment of Anti-human Aggrus Monoclonal Antibody—We generated a mAb to hAggrus (YM-1) as described in
the previous section. The reactivity of YM-1 was confirmed byimmunohistochemical analysis, Western blot analysis, andflow cytometric analysis (Fig. 1, A–C). YM-1 clearly recognizedlymphatic vessel via immunostaining (Fig. 1A). Approximately
36 kDa of Aggrus expressed on human lymphatic endothelial
cells and lung cancer cell line NCI-H226 were detected by YM-1
but not anti-podoplanin mAb (Fig. 1B). This result indicatedthat the YM-1 exhibit more intense immunoreactivity for Aggrus than that of the anti-podoplanin mAb. Furthermore, flow
cytometric analysis showed that the YM-1 could react morestrongly than anti-podoplanin (Fig. 1C).
Platelet Aggregation by Lymphatic Endothelial Cells and
NCI-H226 Cells—As shown in Fig. 1D, lymphatic endothelial
and NCI-H226 cells also induced platelet aggregation as hAggrus
on CHO cells did. However, another lung cell carcinoma NCIH520, which did not express hAggrus, did not induce platelet
aggregation (data not shown). These results indicated that the
spontaneous hAggrus also induced platelet aggregation.
Platelet Aggregation by Lec Transfectants—Recombinants
were generated in the three mutant cells and parental CHO
cells by transfection of pcDNA-hAGR and pcDNA-mAGR toassess the physiological importance of glycosylation on Aggrus
molecules. The stable transfectants were established by the
Geneticin-selective culture. Expression of recombinant molecules was comparatively confirmed by flow cytometry using thespecific antibodies of YM-1 and 8F11 (Fig. 2A). Judging fromthe shift of peaks, expressional levels of Aggrus were high andequal among these cell transfectants.
When the platelet aggregation activity of the establishedtransfectants was examined, both hAggrus and mAggrus in
Lec1 cells induced complete platelet aggregation as well asrecombinants in parental CHO cells (Fig. 2B). The aggregation
was confirmed using varying cell numbers in three independent experiments (data not shown). Real features of induced
platelet aggregation are also demonstrated in Fig. 2C as a

FIG. 3. Lectin blot analysis to immunoprecipitated human Aggrus. Immunoprecipitated hAggrus was applied to SDS-PAGE. The
transferred membranes were incubated with YM-1 (A) and lectins including WGA (B), s-WGA (C), Jacalin (D), peanut agglutinin (PNA) (E),
VVA (F), Ricinus communis agglutinin I (RCA120) (G) and Griffonia
simplicifolia lectin I (GSL-I) (H). Lane 1, CHO-hAggrus cells; lane 2,
lec1-hAggrus cells; lane 3, lec2-hAggrus cells; lane 4, lec8-hAggrus cells;lane 5, parental CHO cells as a negative control. WB, Western blotting;
LB, lectin blotting.

photograph. In contrast, aggregation was absent in Aggrus
molecules expressed in Lec2 and Lec8 cells.
Lec1 CHO cells are incapable of synthesizing N-glycan
complexes (18 –20). The amino acid sequence of mAggrus
contained one consensus site for N-glycosylation but did not
in hAggrus. Because Lec1 cells lacked N-glycosylation, platelet aggregation activity was not associated with this type ofmodification. Furthermore, CMP-SA transporter-deficient
Lec2 cells lack 90% of common SA decoration in both glycoproteins and glycolipids (21). Maturation of sialylated structures is inhibited in Lec8 cells, because Gal is the major
acceptor for SA in glycoconjugates (22, 23). Molecules thatare modified by these two cell lines form insufficient SA
decoration. Based on these results and interpretation, thepresence of SA on O-glycans was conclusively critical for the
induction of platelet aggregation.
Glycans on Aggrus Expressed in CHO and Lec Cells—To typethe O-glycan structure on active hAggrus molecules in the
CHO cells, lectin blotting using the 18 lectins, was further
performed with immunoprecipitates of the cell lysates (Fig. 3).
Table I summarizes this result. The hAggrus that was expressed in Lec1 and parental CHO cells reacted with WGA but
not s-WGA. The aggregation-inducing molecules carried SA attheir termini because s-WGA recognizes naked GlcNAc in contrast to the interaction of WGA to SA-bound GlcNAc. Jacalin,
which recognizes core 1 structure (Gal␤1,3GalNAc), reactedwith hAggrus molecules expressed in parental, Lec1, and Lec2
CHO cells. This result implies that active molecules possess asialylated core 1 structure. Furthermore, hAggrus from Lec 8

38842Aggrus Activity Is Regulated by Glycosylation

TABLE I
Summary of lectin blot analysis to immunoprecipitated hAggrus
For concanavalin A, D. biflorns agglutinin, S. japonica agglutinin, L. esculentum lectin, S. tuberosum lectin, D. stramonium lectin, M. amurensis
lectins I and II, and S. nigra lectin, lectin did not react toward recombinant hAggrus. ⫺, negative; ⫹, positive; w/o, without.
LectinSpecificity

CHO/hAggrus

Lec1/hAggrus

Lec2/hAggrus

Lec8/hAggrus

WGA
s-WGA
Jacalin
Peanut agglutinin
VVA
R. communis agglutinin I
G. simplicifolia lectin
Soybean agglutinin(GlcNAc)n, SA(GlcNAc)nGal␤1,3GalNAc
Gal␤1,3GalNAc (w/o SA)
GalNAc␣, ␤Gal, GalNAc (w/o SA)
GalNAc␣, Gal␣
GalNAc␣, ␤

⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫺

⫹
⫺
⫹
⫺
⫹
⫺
⫺
⫺

⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫺

⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫹

FIG. 4. Scheme representing a possible pathway for O-glycans of Aggrus expressed in Lec mutant cells. The initiating structure of
O-glycan is the addition of the monosaccharide GalNAc to serine and threonine residues (Tn antigen). Subsequently, particular glycosyltransferases form T or sialyl Tn. Possible glycan structures formed by Lec mutant cells appear at intermediate products. Lectins that recognized theoligosaccharide were shown in parentheses.

cells was detected by VVA (to GalNAc-Ser/Thr, called Tn-antigen), Griffonia simplicifolia lectin I, and soybean agglutinin (toGalNAc residue), indicating that the major glycan on hAggrus
from Lec8 was Tn-antigen. In addition, hAggrus moleculesfrom Lec2 cells showed slower movement in electrophoresis
compared with others. This lower mobility might result fromthe reduction of negative charges or elongation of O-glycan
chain because of the lack of SA. Fig. 4 schematically illustrates
the theoretically possible glycosylation structures of Aggrus
expressed in the parental CHO and Lec mutant cells.
DISCUSSION

We generated anti-hAggrus mAb by immunizing rats with thesynthetic Aggrus peptide. Recently, Aggrus/podoplanin has beenutilized as a lymphatic endothelium marker in histopathology.
The results of YM-1 immunostaining indicated that this antibody
has superior sensitivity to lymphatic endothelium (Fig. 1A).
Western blot and flow cytometric analyses indicated that thehAggrus protein is expressed in the lymphatic endothelial cellline and lung cancer cell line, NCI-H226 (Fig. 1, B and C). Furthermore, these results showed that the YM-1 antibody reacted
more strongly than the anti-podoplanin antibody.
The present study utilized the advantage of unique characteristics of CHO mutant cell lines to show the sialylated Oglycan was critical for the platelet aggregation-inducing activity of Aggrus. O-Glycan profiles of MUC1 expressed in CHO celllines showed an identical pattern to those produced in thebreast carcinoma T47D cells (27), suggesting that recombinantAggrus Activity Is Regulated by Glycosylation
Aggrus in CHO cells, which provoked platelet aggregation,
might be decorated as well as glycosylation in cancer cells.
Although the physiological importance of the platelet-aggregating activity remains obscure, the fact that Aggrus is expressedin many tumor cells indicated that cancerous glycosylation of
Aggrus is attributable to the tumor-induced activity of platelet
aggregation during metastasis (3, 6).
Lectin blot analysis showed that active Aggrus molecules
expressed in CHO cells bore a sialylated core 1 structure (SAGal␤1,3GalNAc). This structure was basically consistent withthose reported in the PA2.26 antigen (28) and gp40 (17). In
addition to sialylated and non-sialylated GalNAc-Ser/Thr,called sTn and Tn antigen, respectively, sialylated and nonsialylated core 1 structures are also known as tumor-associatedcarbohydrate antigens (29, 30). These antigens are distributed
widely in glycolipids, membranous mucins, and mucous plasmacomponents in cancer patients. MUC1 mucin has been studied
extensively as a target antigen for the immunotherapy ofbreast and other carcinomas, because the cancer-associatedmucin is over-expressed and aberrantly glycosylated. In normal cells, O-glycans expressed on MUC1 typically consist oflong and branched, mainly core 2 (GlcNAc␤1– 6(Gal␤1–3)GalNAc-Ser/Thr)-based, oligosaccharides (31, 32). In contrast, theglycans of MUC1 expressed in breast carcinomas transform toincreased contents of the shorter non-branched core 1-based
structure with enhanced SA addition (33–35). From this pointof view, Aggrus in carcinoma cells is thought to be distinct fromT1␣/podoplanin in normal cells in glycosylation. However, Aggrus in lymphatic endothelial cells and Aggrus in NCI-H226
showed almost the same mobility in Western blot analysis, andboth cells induced platelet aggregation (Fig. 1B). The different
mobilities of Aggrus from different organs were indicated inprevious studies with mouse Aggrus/PA2.26 (3, 28). The different mobilities may indicate different glycosylation or conformational changes that were caused by different modifications.
Further studies are necessary to analyze the glycan structure
on Aggrus molecules from cancer cells and normal cells, especially the glycan in the platelet aggregation-stimulating domain, to clarify whether the different glycosylation has animportant correlation with the platelet aggregation activity ofAggrus or not.
Recent progress in research has demonstrated that the glycosyl modification is relevant to various physiological functions
such as the intracellular signaling pathway (36). Notch functions in highly conserved intercellular signaling pathways. It
plays essential roles in a wide variety of developmental events.
A series of studies with deficient CHO cells, including Lec2 andLec8, have been applied for the Fringe-dependent glycosylationof Notch (36, 37). Fringe is fucose-specific ␤1,3 N-acetylglucosaminyl transferase that adds GlcNAc to O-fucose on certain
epidermal growth factor repeats of Notch (36, 38). The change
in sugar structure affects ligand binding (38, 39).
Similarly, exploiting deficient CHO cells, we found that sialylated glycans on Aggrus promotes platelet aggregation. It is
intriguing to speculate that the change in glycosylation modification affect platelet aggregation-inducing activity of Aggrus
in vivo. Based on our findings that sialylated glycans on Aggrus
induced platelet aggregation, Aggrus interacted with a ligandon the platelet surface. We have preliminarily investigated theassociation of Aggrus with three SA-binding Ig-like lectins(Siglec) of Siglec-3, Siglec-5, and Siglec-7, which are expressedin platelets (40). However, no positive evidence has been obtained by Western blotting and flow cytometry to date (data notshown). In the next step, it will be important to determine

38843

which acceptant component on platelets is attributable to thepotent activity toward aggregation by Aggrus.
In conclusion, we generated a new anti-hAggrus antibody,YM-1. Then we showed that the decoration of SA on O-glycan isessential for platelet aggregation by membranous Aggrus molecules. Such data seem particularly important in light of thefact that the platelet aggregation activity is related to tumor
metastasis.
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