Gene 378 (2006) 52 – 57
www.elsevier.com/locate/gene

Conservation of a platelet activating domain of Aggrus/podoplanin
as a platelet aggregation-inducing factor
Mika Kato Kaneko a,b,⁎, Yukinari Kato a,b , Takashi Kitano a , Motoki Osawa a,cab

Department of Experimental and Forensic Pathology, Yamagata University School of Medicine, 2-2-2, Iida-nishi, Yamagata 990-9585, Japan
Glycogene Function Team of Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), OSL C2, 1-1-1,
Umezono, Tsukuba 305-8568, Japanc
Department of Forensic Medicine, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-1193, Japan
Received 7 January 2006; received in revised form 22 April 2006; accepted 25 April 2006
Available online 11 May 2006
Received by N. Saitou

Abstract
Human Aggrus/podoplanin is an identified platelet aggregation-inducing factor of cancer cells, which is also known as a specific marker of lymphatic
endothelium. Human Aggrus was known to be expressed in seminoma, squamous cell carcinoma, malignant mesothelioma, sarcomas and several brain
tumors. In our previous studies, the sialylated O-glycan of human and mouse Aggrus were shown to be critical for its platelet aggregation-inducing activity
in the experiments using the glycosylation-deficient Chinese hamster ovary (CHO) cell lines. We newly cloned Aggrus homologues from rat, hamster, dog
and bovine cDNAs, in addition to the human and mouse cDNAs, and confirmed there are three tandem repeats of the platelet aggregation-stimulating(PLAG) domain in Aggrus, which were conserved in all homologues. We found that bovine Aggrus has a sporadic deletion mutation in the first PLAG
domain, and lacks platelet aggregation-inducing activity. We introduced point mutation in the PLAG domain of Aggrus and showed that either the first or
last PLAG domain is critical for activity, but not the middle domain. In addition, we studied the molecular evolutionary process of the PLAG domain ofAggrus. The PLAG domain and its activity appeared after the divergence of avians and mammals. In conclusion, we provide evidence that Aggrus
homologues conserved the segment of EDxxVTPG in their extracellular domain which are critical for their platelet aggregation-inducing activities.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Platelet aggregation; PLAG domain; Homologous gene

1. Introduction
Platelet aggregation is known to be related to cancer metastasis.
Our previous studies have clarified that membranous 44-kDa and
36-kDa sialoglycoproteins, respectively of cancer cells of mice andhumans, aggregated platelets with no relation to plasma components (Kato et al., 2003; Kaneko et al., 2004). Ectopic expression ofAbbreviations: CHO, Chinese hamster ovary; PLAG, platelet aggregationstimulating; PCR, polymerase chain reaction; RT, reverse transcriptase; ORF,
open reading frame; PRP, platelet-rich plasma; PBS, phosphate buffered saline;
SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence kit; ERM,
ezrin, radixin, moesin; WT, wild type; EST, expressed sequence tag; HMM,
Hidden Markov Model; PAGE, polyacrylamide gel electrophoresis.
⁎ Corresponding author. Glycogene Function Team of Research Center for
Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), OSL C2, 1-1-1, Umezono, Tsukuba 305-8568, Japan. Tel.: +81 29
861 3197; fax: +81 29 861 3191.
E-mail addresses: mk-kaneko@aist.go.jp, mikanekok@hotmail.com(M.K. Kaneko).
0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2006.04.023

these molecules, designated as Aggrus, was observed in various
cancer cells, including testicular tumors, squamous cell carcinomas, mesothelioma, and brain tumors, in which up-regulated expression was evident (Kato et al., 2003, 2004, 2005; Kimura andKimura, 2005; Martin-Villar et al., 2005; Mishima et al., 2006a,b;
Wicki et al., 2006). Unique characteristics of Chinese hamster
ovary (CHO) mutant cell lines Lec1, Lec2 and Lec8 revealed thatsialylated O-glycan is critical for platelet aggregation-inducing activity (Kaneko et al., 2004).
Homologous molecules to Aggrus have been identified independently in several mammals: T1α (Dobbs et al., 1988; Rishi
et al., 1995), podoplanin (Breiteneder-Geleff et al., 1997) andPA2.26 (Gandarillas et al., 1997) are isolated from rat alveolar typeI cells in lung, rat glomerular epithelial cells (podocytes) or mouse
keratinocytes. Other homologues include the canine receptor for
the influenza C virus gp40 (Gandarillas et al., 1997) and its humanhomologue gp36 (Zimmer et al., 1999) and RANDAM-2 (Kotani
et al., 2003), the membrane glycoprotein expressed in neuronalM.K. Kaneko et al. / Gene 378 (2006) 52–57

53

cells during mouse neurogenesis. Podoplanin is used as a specificmarker for lymphatic endothelium in histopathology (Breiteneder-Geleff et al., 1999). Ramirez et al. showed that T1α null mice,
generated by a targeted dysfunction of the gene, died at birth oflethal respiratory failure accompanied by immature lymphatic
vessel formation (Ramirez et al., 2003; Schacht et al., 2003).
Furthermore, T1α/podoplanin conceivably plays an importantrole in regulating peripheral lung cell proliferation and lymphatic
vascular development.
In the present study, we cloned aggrus homologues from rat,
hamster, dog and bovine cDNAs, in addition to human and mouse
aggrus, and examined platelet aggregation-inducing activity. All
Aggrus homologues, except for the bovine homologue, showed
platelet aggregation-inducing activity. We also conducted evolutionary analysis of the aggrus genes.

geneticin (G418; Sigma-Aldrich, St. Louis, MO) at a concentrationof 1.0 mg/ml for a couple of weeks. Human, mouse and mocktransfectants were transiently obtained.

2. Materials and methods

Cultured cell pellets were lysed with a cell lysis buffer (25 mM
Tris, 50 mM sodium chloride, 0.2% sodium dodecyl sulfate (SDS),
0.5% sodium deoxycholate, 2% nonidet P-40, 50 μg/ml aprotinin,and 1 mM phenyl methylsulfonyl fluoride; it was pH 7.4) for
30 min on ice. Western blotting was performed as described previously (Kaneko et al., 2004). For detection of rat and dog Aggrus
combined with His tag were performed with anti-His tag antibody(Medical Biological Laboratories Co., Ltd., Nagoya, Japan),whereas hamster and bovine Aggrus with FLAG-tag were done byan anti-FLAG M2 antibody (Sigma-Aldrich). The blotting results
were developed using an enhanced chemiluminescence kit (ECL,
Amersham biosciences corp., Piscataway, NJ).

2.1. cDNA cloning of aggrus genes and induction of point
mutations
The cDNA encoding the full-length open reading frame (ORF)of rat and dog aggrus was obtained by PCR using rat lung cDNA
(OriGene Technology Inc., Rockville, MD) and cDNA derived
from the Madin-Darby Canine Kidney cell line (American Type
Culture Collection) as templates (GenBank Accession Nos.
U96449 and Z81018). We designed degenerate primers with reference to alignment of those aggrus genes (human, mouse, rat anddog) to obtain other mammalian aggrus genes. The primer set for
aggrus was as follows: HindIII-AGR-dF1, 5′-ccaagcttATGTGGAVSGYGYCAGYBYTGYTCT-3′ and EcoRI-AGR-dR487 5′gggaattcGGGCGAGWACCTTCCHGAMATYTT-3′. Total RNA
was extracted from a hamster: testis; a bovine: lung, colon andmuscle. The RNA was isolated from each organ using a QIAGEN
RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The accession numbers of hamster andbovine aggrus sequences are AB205159 and AB205160, respectively. To assess the platelet aggregation-inducing activity, the fulllength cDNAs of human, mouse, hamster and bovine aggrus, incorporated with FLAG-tag sequence, were subcloned into
pcDNA3 vector (Invitrogen Corp., Carlsbad, CA), or rat and dog
aggrus were subcloned into pcDNA3.1/V5-His-TOPO (Invitrogen). Substitution of the appropriate amino acid codons to alanine
codons in aggrus cDNAs was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
2.2. Recombinant aggrus expression on CHO cell lines
CHO cells were cultured at 37 °C in a humidified atmosphere of
5% CO2 in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum (Biocell, Carson, CA), 2 mM Lglutamine (Invitrogen), 100 U/ml of penicillin (Banyu pharmaceutical, Tokyo), and 100 μg/ml of streptomycin (Meiji-Seika,
Tokyo). Constructed vectors were transfected into CHO cells by thelipofectamine method (Invitrogen) according to the manufacturer's
instruction. For rat, hamster, dog and bovine aggrus, stable transfectants were obtained using a selective culture medium containing

2.3. Platelet aggregation
Mouse platelet-rich plasma (PRP) was prepared from fresh,
heparinized blood extracted from BALB/c mice. An aliquot of
200 μl of PRP was incubated in a cuvette at 37 °C under continuous
stirring. After 5 min, 5 μl of phosphate buffered saline (PBS)washed cells were added. Then light transmittance was monitored
at 660 nm for 15 min. This assay was performed with NKK HEMA
Tracer I (Niko Bioscientific Co., Tokyo, Japan).
2.4. Western blot analysis

2.5. Molecular evolutionary analysis
Multiple alignment was constructed by the CLUSTAL W program (Thompson et al., 1994), and illustrated with MacBoxshade,
which was also used to calculate sequence identity and similarity. All
Aggrus sequences were analyzed using the NetOGlyc neural network for the identification of O-linked glycosylation sites (http://
www.cbs.dtu.dk/netOglyc/) (Hansen et al., 1998; Julenius et al.,2005). For constructing a phylogenetic tree, we assumed three topologies as follows: topology 1; ((((mouse, rat), hamster), bovine),human, dog), topology 2; ((((mouse, rat), hamster), human), dog,
bovine), and topology 3; ((((mouse, rat), hamster), dog), human,
bovine), where rodents form a cluster for all three topologies. The
maximum likelihood method with JTT model (Jones et al., 1992)
was used to compare these topologies. The CODEML program foramino acid sequence data in the PAML package (Yang, 1997) wasused for the estimation. Although the topology 1 showed the highest
maximum likelihood value (−1675.628), this is not statistically significant from topologies 2 (−1678.189±3.564) and 3 (−1676.649±
4.114). Thus, we used topology 2 as a plausible tree, because humanand rodents (both belong to Euarchontoglires) form a cluster.
3. Results
3.1. Cloning and sequencing of aggrus homologues
We previously subcloned human and mouse aggrus genes(Kato et al., 2003). In this study, we newly subcloned and seqenced

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M.K. Kaneko et al. / Gene 378 (2006) 52–57

rat, hamster, dog and bovine aggrus. Fig. 1A showed the multiplealignments of six amino acid sequences of Aggrus. At the aminoacid level, identity was determined between human and rodents(means 46%), human and dog (64.5%) or human and bovine(38.6%). Although overall homology was poor, the PLAG domain

and transmembrane domain were highly conserved. Fig. 1B showsa schematic representation of the Aggrus protein structure. All
Aggrus homologues belong to a type-I transmembrane sialomucin-like glycoprotein that consists of an extracellular domain withthree repeats of the PLAG domain, abundant serine and threonine

Fig. 1. Comparison of the amino acid sequences of various Aggrus homologues. (A) Multiple alignment of the amino acid sequences. Completely conserved residues
are shaded black; conserved substitutions are shaded gray. (B) Schematic representation of the PLAG domain, transmembrane and the predicted glycosylation sites ofAggrus. The PLAG domain is indicated as a box. Box pattern indicated the degree of PLAG domain homology.

M.K. Kaneko et al. / Gene 378 (2006) 52–57

residues as potential O-glycosylation sites, a single transmembrane
portion and a short cytoplasmic tail. The cytoplasmic tail contained
potentially binding site of ezrin, radixin, moesin (ERM) protein
and potential protein kinase A/protein kinase C-dependent phosphorylation serine residue (Martin-Villar et al., 2005). Bovine

55

Aggrus lacks the potential phosphorylation serine residue. Using
NetOGlyc 3.1, we predicted a potential O-glycosylation site ofthese Aggrus sequences. The threonine residues in the PLAG domain were inferred to be O-glycosylation sites. Subsequent sequences contained a high proportion of potential O-glycosylation

Fig. 2. Platelet aggregation-inducing activity of transfected cells. Mouse platelet-rich plasma (PRP) was prepared from fresh, heparinized blood extracted from BALB/
c mice. An aliquot of 200 μl of PRP was incubated in a cuvette at 37 °C under continuous stirring with transiently transfected (A) human Aggrus WT, T52A; (B) mouse
Aggrus WT, T34A; (C) mock transfectant, or with stably transfected (D) rat Aggrus WT, T34A; (E) hamster Aggrus WT, T34A, T43A; (F) dog Aggrus WT, T41A,
T50A; (G) bovine Aggrus WT. Then light transmittance was monitored at 660 nm for 15 min. This assay was performed with NKK HEMA Tracer I. Transfected cellpellets were lysed with a cell lysis buffer for 30 min on ice. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 10% constant gels. After proteins
were transferred onto immobilon polyvinylidene difluoride membrane, the strips were incubated with anti-His tag antibody for rat and dog Aggrus, or with FLAG-tag
antibody for hamster and bovine Aggrus. The blotting results were developed using an enhanced chemiluminescence kit.

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M.K. Kaneko et al. / Gene 378 (2006) 52–57

serine and threonine residues. However, neither the exact number
nor the positions of glycosylated residues seem to be conserved
among human, rodents, dog and bovine Aggrus.
3.2. Confirmation of platelet aggregation-inducing activity ofAggrus homologues
Expression of recombinant molecules was confirmed byWestern blot analysis in Fig. 2. We previously found out theplatelet aggregation-inducing activity of human and mouse
Aggrus (Fig. 2A and B; Kato et al., 2003). In this study, wild
type (WT) of rat, hamster and dog Aggrus, which were expressed on CHO cells, respectively, induced platelet aggregation in the same way (Fig. 2D, E and F). However, wild typebovine Aggrus did not induce platelet aggregation (Fig. 2G).
The bovine Aggrus has a sporadic deletion mutation in aminoacid sequence of the PLAG domain (refer to Fig. 1).
Previously, we generated point mutants of human-T52A andmouse-T34A Aggrus, which did not induce platelet aggregation-inducing activity (Fig. 2A and B; Kato et al., 2003). In thisstudy, we newly generated several point mutants of aggrus
homologues. Mutant Aggrus (rat-T34A, hamster-T34A anddog-T41A) did not induce platelet aggregation, but mutant
hamster (hamster-T43A) and dog Aggrus (dog-T50A) did (Fig.
2D, E, and F). These results indicated that the most conserved
PLAG domain, either the first or the last, was critical for activity, whereas the middle one did not affect platelet aggregationinducing activity.
3.3. Molecular evolutionary analysis
We constructed a phylogenetic tree of six mammalian Aggrus
amino acids sequences (Fig. 3). The rodents and the bovine lineages showed longer branches in contrast with the human and the
dog lineages. The longer branch of rodents results from higher
rates of amino acid substitution in rodents (Gu and Li, 1992). It is
likely that some kinds of functional changes are related to higher
rates of amino acid substitution in the bovine lineages. In fact,
bovine Aggrus did not induce platelet aggregation, as shown in
Fig. 2G, probably because of a sporadic deletion mutation inamino acid sequence of the PLAG domain.

Fig. 3. A phylogenetic tree of aggrus genes. Rates of amino acid substitutions were
calculated by using JTT model with gamma correction. An estimated gamma shapeparameter (α = 1.6) was used for the gamma distribution. A root was not definedand showed by trichotomy style of bovine, dog, and human–rodents.

Furthermore, we found a similar gene in chicken expressedsequence tag (EST) database using the TBLASTN algorithm. A
putative chicken orthologue had neither a PLAG domain nor Oglycosylated sites (data not shown). It suggests that the PLAG
domain arose and was triplicated before the appearance of mammals. Speciation of mammals might have occurred after thedomain was triplicated. Then, a deletion mutation of the first
PLAG domain happened in the bovine lineage (Fig. 3).
4. Discussion
We cloned rat, hamster, dog and bovine aggrus, in addition topreviously cloned human and mouse aggrus. Human, rodents anddog Aggrus exhibit similar structural and biochemical characteristics and likely share an identical function: platelet aggregationinducing activity. Unexpectedly, bovine Aggrus showed no activity. The introduced point mutations were located in the last
PLAG domain for human Aggrus and the first PLAG domains forrodents and dog Aggrus, which were the most conserved part
among three PLAG domains. Furthermore, we introduced point
mutation in hamster-T43A (the middle domain) and dog-T50A(the middle domain). Human-T52A, mouse-T34A, rat-T34A,
hamster-T34A and dog-T41A showed no platelet aggregationinducing activity, but hamster-T43A and dog-T50A did. These
results indicated that the most conserved PLAG domain is criticalfor activity. However, the middle PLAG domain did not affecttheir activity, even though the PLAG sequence is highly conserved. It will be interesting to investigate the tertiary structure of
the three tandem PLAG domains, which might yield important
information for exploitation in a novel anti-coagulant.
Fig. 1B shows that the first (rodents, dog and bovine) or the last(human and dog) PLAG domain was highly conserved. The
middle domain seems to be released from evolutional pressure.
For that reason, mutations were accumulated in the middle domain. However, in the case of rodents and dog Aggrus, the middle
PLAG domain was also conserved: it is speculated to be overwritten with the sequence of first PLAG domain by gene conversion or crossing-over.
Using the TBLASTN algorithm, we found that mouse Homeobox protein Nkx-3.2 has a PLAG sequence — EDxxVTPG.
An evolutionary conserved homologue of the Drosophila bagpipe
homeobox gene, Nkx-3.2, was expressed in the splanchnic mesoderm and the embryonic skeleton. Furthermore, using profile
Hidden Markov Model (profile HMM) method, we found a
PLAG domain-containing sequence in Neurocan. The profile
HMM method turns a multiple sequence alignment into a position-specific scoring system that uses databases to search forremote homologous sequences (Eddy, 1998). Neurocan is a coreprotein of a chondroitin sulfate proteoglycan; it is abundant infetal brain tissue. It is inferred to be involved in modulation of celladhesion and migration. Both Nkx-3.2 and Neurocan seem to beunrelated to platelet aggregation-inducing activity. We think thatthe platelet aggregation-inducing activity is not the only functionof Aggrus. This newly identified PLAG domain might have anovel biological activity.
To our knowledge, this is the first detailed study of Aggrus
PLAG domain. There are three tandem repeats of PLAG domain,M.K. Kaneko et al. / Gene 378 (2006) 52–57

EDxxVTPG, in their extracellular domain of Aggrus/podoplaninhomologues. Either the first or last PLAG domain is critical for
activity, but not the middle domain. Furthermore, we studied themolecular evolutionary process of PLAG domain of Aggrus. It
would lead us to understand the mechanism of the platelet aggregation, and help us to exploit anti-coagulant therapies.
Acknowledgement
We thank Drs. Fujita and Tsuruo for their great help, Ms.
Kunita and Mr. Nakazawa for their kind assistance and Dr.
Shikanai for skillful technical help of profile HMM analysis. We
owe thanks to Ms. Kugota for great help through work. This
study was supported in part by a Grand-in-Aid for Scientific
Research, 17770082, from the Ministr of Education, Culture,
Sports, Science and Technology of Japan (to M.K.K.), Kanae
Foundation for Life and Socio-medical Science (to Y.K.), andOsaka Cancer Research Foundation (to Y.K.).
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