Biochemical and Biophysical Research Communications 533 (2020) 57e63

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Crystal structure of an anti-podoplanin antibody bound to adisialylated O-linked glycopeptide
Satoshi Ogasawara a, b, *, Kano Suzuki a, Kentaro Naruchi c, Seiwa Nakamura a,Junpei Shimabukuro c, Nanase Tsukahara c, Mika K. Kaneko d, Yukinari Kato d, e,Takeshi Murata a, b, **a

Graduate School of Science, Chiba University, Chiba, Japan
Molecular Chirality Research Center, Chiba University, Chiba, Japan
Medicinal Chemistry Pharmaceuticals, Co., Ltd., Sapporo, Japand
Tohoku University Graduate School of Medicine, Sendai, Japane
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 26 August 2020
Accepted 27 August 2020
Available online 10 September 2020

Podoplanin (PDPN) is a highly O-glycosylated glycoprotein that is utilized as a speciﬁc lymphatic
endothelial marker under pathophysiological conditions. We previously developed an anti-human PDPN
(hPDPN) monoclonal antibody (mAb), clone LpMab-3, which recognizes the epitope, including both the
peptides and the attached disialy-core-l (NeuAca2-3Galbl-3 [NeuAca2-6]GalNAcal-O-Thr) structure at
the Thr76 residue in hPDPN. However, it is unclear if the mAb binds directly to both the peptides andglycans. In this study, we synthesized the binding epitope region of LpMab-3 that includes the peptide(-67LVATSVNSV-T-GIRIEDLP84-) possessing a disialyl-core-1 O-glycan at Thr76, and we determined the
crystal structure of the LpMab-3 Fab fragment that was bound to the synthesized glycopeptide at a 2.8 Å
resolution. The six amino acid residues and two sialic acid residues are directly associated with four
complementarity-determining regions (CDRs; H1, H2, H3, and L3) and four CDRs (H2, H3, L1, and L3),
respectively. These results suggest that IgG is advantageous for generating binders against spacious
epitopes such as glycoconjugates.
© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Keywords:
Podoplanin (PDPN)
Monoclonal antibody
Crystal structure
Glycopeptide

1. Introduction
Podoplanin (PDPN) was previously discovered in renal podocytes [1], and it was recently reported as a novel biomarker ofischemia-reperfusion injury (IRI), as an increase in the urine PDPNto-creatinine ratio is strongly correlated with the onset of renal IRI[2]. PDPN has also been reported to be expressed in lymphatic
endothelial cells, but not in vascular endothelial cells [3,4]. Basedon this, PDPN can be utilized as a speciﬁc lymphatic endothelial
marker under pathophysiological conditions [5]. PDPN binds to theC-type lectin-like receptor-2 (CLEC-2) of platelets to induce platelet* Corresponding author. Graduate School of Science, Chiba University, Chiba,
Japan.
** Corresponding author. Graduate School of Science, Chiba University, Chiba,
Japan.
E-mail addresses: satoshi-ogasawara@chiba-u.jp (S. Ogasawara), t.murata@
faculty.chiba-u.jp (T. Murata).

aggregation [6,7]. PDPN was therefore previously referred to as“Aggrus” [8]. PDPN-CLEC-2 signaling leads to platelet aggregation, aprocess that is critical for embryonic blood-lymphatic vascularseparation [9]. PDPN is also abundant in lung type I alveolar cells,
where it is called as “T1aˮ [10]; however, it is not expressed in type
II alveolar cells. Based on the use of PDPN as a speciﬁc marker for
type I alveolar cells, the development and function of type I alveolar
cells has been investigated in detail [11].
PDPN is also overexpressed in various tumors, including oral
cancer, lung cancer, esophageal cancer, malignant brain tumors,
mesotheliomas, testicular tumors, and osteosarcoma [8,12e21].
Number of reports have indicated that PDPN expression is associated with malignant progression and cancer metastasis [7,17], and
PDPN expression is also associated with clinical outcomes [22].
PDPN has also been reported to be expressed by tumor-initiating
cells (TICs) that are believed to be responsible for tumor relapse
and resistance to conventional therapies [23].
25 years ago, Toyoshima et al. reported that mouse PDPN/gp44

https://doi.org/10.1016/j.bbrc.2020.08.103
0006-291X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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is highly O-glycosylated and that this glycosylation is associatedwith platelet aggregation [24]. In our previous studies that examined the effect of glycosylation on platelet aggregation activity,
recombinant podoplanins were expressed by Lec1 (lacking Nglycan), Lec2 (lacking sialic acid) and Lec8 (lacking galctose), suchas glycosylation mutants of Chinese hamster ovary (CHO) cell [25].
PDPN treatment of Lec1 cells induced platelet aggregation; however, this was not observed in Lec2 and Lec8 cells. Lectin blot analyses suggested that sialylation of core-1 structures were criticalfor platelet aggregation activity [25]. Thus the activity was affectedby the sialic acid and revivable by additional sialylation [26].
Additionally, a further analysis clariﬁed that the Thr52 of human
PDPN was glycosylated the disialyl-core-l (NeuAca2-3Galbl3(NeuAca2-6)GalNAcal-O-Thr) structure [26]. These results indicated that the sialylated core-l at Thr52 of human PDPN is criticalfor PDPN-induced platelet aggregation. To elucidate the structural
basis of the glycan-dependent interactions between PDPN and
CLEC-2, we performed crystallographic studies of PDPN in complex
with CLEC-2 [27]. The interaction of human PDPN with CLEC-2
primarily involves Glu47 and Asp48 in the platelet aggregationstimulating domain-3 (PLAG3) and the a-2, 6 linked sialic acidattached on Thr52 of human PDPN [27]. Interestingly, this PLAG
domain is highly conserved among PDPNs of various species [28].
PLAG domains such as PLAG1, PLAG2, and PLAG3 are located within
the N-terminus of PDPN [28], and PLAG-like domains (PLDs), also
named PLAG4 [29], are found in the middle of PDPNs [30]. Although
the disialyl-core-l structure is attached within PLAG domains and
PLDs and is primarily involved in PDPN-CLEC-2 interaction [7,29],
the other Ser/Thr residues in PDPN may also possess O-glycans, as
half of the molecular weight of PDPN is known to be derived fromglycans [26]. The function of these O-glycans has not been clariﬁed.
We have previously developed a number of monoclonal antibodies (mAbs) against human PDPN [15,31]. The binding epitopesof several anti-human PDPN mAbs were determined to include
both peptides and glycans, including LpMab-2, LpMab-3, LpMab-9,
LpMab-12, LpMab-19, and LpMab-21 [31e36], that were developed
using Cancer-speciﬁc mAb (CasMab) technology [31]. We named
this type of mAbs as anti-glycopeptide mAbs (GpMabs). Epitope
mapping is typically performed using the alanine scanning method,
and the binding epitope of LpMab-2 includes Thr55/Ser56, while
the binding epitopes of LpMab-3, LpMab-19, and LpMab-21 include
Thr76, that of LpMab-12 includes Thr52, and that of LpMab-9 includes Thr25. However, we have not clearly demonstrated if

GpMabs can directly react with both peptides and glycans. In thisstudy, we ﬁrst synthesized the PDPN-glycopeptide that included
Thr76. We determined the crystal structure of a complex of LpMab3-Fab and PDPN-glycopeptides, and our results revealed that theantibody directly binds to both peptides and glycans tightly.
2. Materials and Methods
2.1. Synthesis of the PDPN-glycopeptide
The PDPN-glycopeptide was synthesized in combination with asolid phase method and glycosyl transferase reaction. Thesemethods were described in detail in Supporting Information.
2.2. Puriﬁcation and crystallization of the Fab fragment
The papain (Nacalai Tesque, Inc., Kyoto, Japan) was immobilizedon NHS-activated Sepharose 4 Fast Flow (GE Healthcare, Uppsala,Sweden). Puriﬁed LpMab-3 IgG was incubated with the immobilized papain in the presence of 2 mM cysteine-HCl and 20 mM
EDTA in phosphate-buffered saline (PBS, pH 7.4) for 8 h at 37  Cunder gentle agitation. After removing the immobilized papain, thereaction mixture was applied onto a pre-equilibrated rProtein A
Sepharose Fast Flow column (GE Healthcare) with PBS containing
2.5 M NaCl. The ﬂow-through fraction containing the Fab fragment
was collected and concentrated, and this was further puriﬁed by
performing gel ﬁltration chromatography on a Superdex 200 10/300 column (GE Healthcare) equilibrated with PBS. The fractions
containing the Fab fragment were collected and concentrated to
10e15 mg/mL. Crystallization trials were performed using thesitting-drop vapor diffusion method at 20  C as described elsewhere [37]. The crystals were obtained by mixing 0.1 mL of puriﬁed
sample with 0.1 mL of reservoir solution (0.1 M Tris-HCl [pH 7.5],
0.2 M magnesium-Acetate, and 25% [v/v] PEG-3350), and they werethen soaked in cryoprotectant containing 20% (v/v) glycerol incrystallization buffer for a few minutes prior to ﬂash freezing.
2.3. Co-crystallization of the Fab fragment and the PDPNglycopeptide
The excess PDPN-glycopeptide was added to LpMab-3/Fab(molar ratio ¼ 100:1) and incubated for 1 h at 4  C. The unbound
PDPN-glycopeptide was removed using a 10 MWCO Amicon-Ultra

Fig. 1. Structures of LpMab-3 Fab fragment with/without PDPN-glycopeptide. Sections (A) and (B) show the overall structures of the LpMab-3 Fab fragment crystallized in theabsence and presence of PDPN-glycopeptide, respectively. (C) Magniﬁed top view of (B). The |Fo|-|Fc| electron density maps calculated without the glycopeptide contoured 3.0 s areshown in green (positive) and red (negative). CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions of the antibody are colored in green, violet, blue, yellow, orange, andred, respectively.

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59

Fig. 2. Structure of the PDPN-glycopeptide. (A) Magniﬁed view of the PDPN-glycopeptide within the crystal structure of the glycopeptide-bound LpMab-3 Fab fragment (shown as a
stick model) with a positive electron density map (shown in green) calculated without the glycopeptide contouring 1.5 s. (B) Structural formula of the synthesized PDPNglycopeptide. The highlighted area in green corresponds to that of the positive electron density map in (A).

(Merck Millipore) ultraﬁltration device and then concentrated to
10e15 mg/mL. Crystallization trials were performed using thesitting-drop vapor diffusion method at 20  C. The crystals wereobtained by mixing 0.1 mL of puriﬁed sample with 0.1 mL of reservoir
solution (0.1 M Tris-HCl [pH 7.5], 0.2 M magnesium-Acetate, and
25% [v/v] PEG-3350), and they were then soaked in cryoprotectant
containing 20% (v/v) glycerol in crystallization buffer for a few
minutes prior to ﬂash freezing.
2.4. Structure determination
All X-ray diffraction data sets were collected from a single
crystal at a cryogenic temperature (100 K). For LpMab-3/Fab boundwith/without PDPN-glycopeptide, X-ray diffraction data sets werecollected on a beamline BL-1A (l ¼ 1.1 Å) located at the Photon

Factory (Tsukuba, Japan). The collected data from LpMab-3/Fabbound without PDPN-glycopeptide were processed to 2.1 Å using
XDS [38]. The structure was solved by molecular replacement with
Phaser [39] using the antibody Fab fragment exo Diels-Alderase
inhibitor complex (PDB ID: 1A3L) as a search model. The
collected data from LpMab-3/Fab bound with PDPN-glycopeptide
were processed to 2.8 Å using XDS [38]. The structure was solved
by molecular replacement with Phaser [39] using the DielsAlderase catalytic antibody Fab fragment (PDB ID: 1A4J; chains A
and B) as a search model.
The atomic models were created manually using Coot [40] andreﬁned iteratively using PHENIX [41]. RAMPAGE was used to validate the reﬁned structures [42], and the crystallographic andreﬁnement statistics are summarized in Supplementary Table 1. Ca
atoms were used for the calculation of all RMSD values. LigPlotþ

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Fig. 3. Interactions between PDPN-glycopeptide and CDRs in LpMab-3. Side-view structures of PDPN-glycopeptide-bound Mab-3 Fab fragment corresponding to the red box in
Fig. 3b. The Fab fragment is illustrated as a cartoon model (A) and surface model (B). The glycopeptide is presented as a stick model. The colors of the CDRs correspond to those in
Fig. 1. (C) and (D) are the top-view structures of (A) and (B), respectively.

was used for the analysis of the glycopeptide-antibody interaction[43], and the ﬁgures were prepared using PyMOL (PyMOL Molec€dinger LLC, New York, NY,ular Graphics System, version 2.1. 1, SchroUSA).

3. Results
3.1. Synthesis of PDPN glycopeptide
We synthesized the PDPN-glycopeptide containing disialylcore-1 at the Thr76 residue (Fig. S1) by using a solid phasemethod and enzymatic reaction. To distinguish two sialic acidresidues in glycan, the sialic acid bound to galactose and the otherbound to N-acethylgalactosamine denote as Sia-3 and Sia-4,
respectively (Fig. S2A).
The binding ability of the synthesized PDPN-glycopeptide to the
LpMab-3 antibody was evaluated using biolayer interferometry(BLItz system). The dissociation constant (KD) value was estimatedto be 2.0 ± 0.5  106 M (Fig. S2C), which was approximately 24fold higher than the value estimated when using puriﬁed recombinant PDPN protein (KD: 8.5  108 M) [35]. The lower binding
afﬁnity of the synthesized glycopeptide may be responsible for thehigher ﬂuctuation of the peptide portion. However, the KD value ofthe glycopeptide appears to be sufﬁcient to obtain the crystal
structure of the complex.

3.2. Crystal structures of an antibody (LpMab-3) Fab fragment
bound with/without the synthesized PDPN-glycopeptide
We puriﬁed the Fab fragment of LpMab-3 using a Protein A
column and gel ﬁltration chromatographies after papain treatment(see Materials and Methods), and we crystallized and determinedthe crystal structure at a resolution of 2.1 Å (PDB ID: 7C95;
Table S1). Two Fab fragments were present within the asymmetricunit of the crystal lattice. The structures of the variable (Fv) regions
closely resembled each other (Ca root-mean-square deviation[RMSD] ¼ 0.61 Å). Fig. 1A shows a Fab fragment (chains A and B)
with an F|o| - F|c| electron density map. No signiﬁcant positiveelectron density was observed surrounding the CDR regions. Next,
we crystallized the Fab fragment in the presence of an excessconcentration of the synthesized PDPN-glycopeptide and obtained
an electron density map at a resolution of 2.8 Å (PDB ID: 7C94;
Table S1). This crystal also contained two antibody fragments
within the asymmetric unit. After Fab fragment molecules wereﬁtted into the density, strong positive electron densities that areinterpreted as glycopeptides were observed surrounding the CDRsof the antibodies (Fig. 3B). We modeled eight amino acid residues(VTGIRIED) and the disialyl-core-1 structure of the glycopeptide byguiding the shape of the electron density map (Fig. 2). There was no
signiﬁcant electron density for the N-terminal amino acid residues(LVATSVNS) located upstream of Val75 or the C-terminal aminoacid residues (LP) located downstream of Asp82 in the

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61

Fig. 4. Schematic representation of the interactions between PDPN-glycopeptide and CDRs in LpMab-3. The distances separating the amino acid residues in LpMab-3 Fab and the
PDPN-glycopeptide were analyzed using the Ligplotþ program. (A) Hydrogen bonds are shown as red dashed lines. The bond length cut off is set to 3.5 Å for hydrogen bond. (B) Van
der Waals contacts are represented by the spoked arcs oriented toward the ligand atoms they contact. The range of distances deﬁning van der Waals contacts is 2.9e3.9 Å. The colorsof the CDRs correspond to those in Fig. 1.

glycopeptide, and this suggests that these residues do not bind to
the antibody and are disordered. The observed eight amino acidresidues and glycans are also indicated as the epitope region of
PDPN for LpMab-3 by biochemical epitope mapping using thealanine scanning method [35]. The obtained structures of two Fabfragment molecules bound to the glycopeptide in the asymmetricunit were nearly identical (RMSD ¼ 0.31 Å). Therefore, we used thedata for a Fab fragment (chains AeC) in all of the ﬁgures and in thefollowing discussion.

3.3. Binding interactions of the glycopeptide with LpMab-3
The eight amino acid residues (VTGIRIED) of the glycopeptide
are located at the cavity between CDR-H2 and CDR-H3 that isformed by 11 hydrogen bonds and 22 van der Waals contacts(Figs. 3 and 4). Arg79 and Glu81 residues primarily contribute tothese interactions, while the Thr76 that links the disialyl-core-1
glycan does not interact with the antibody. The disialyl-core-1
glycan is located on the surface of CDRs L3 and H2 that is formedby three hydrogen bonds and nine van der Waals contacts (Figs. 3
and 4). In particular, the a-2, 6 linked sialic acid residue (Sia-4)
contributes to the majority of the interaction with the antibody. The
other sialic acid residue (Sia-3) interacts with CDR-H2 Thr58 andTyr59. The rest of the reduced saccharides (GalNAc-1 and Gal-2) do
not interact with the antibody. Thus, the antibody LpMab-3 directly
associates with six amino acid residues and two sialic acid residues.

4. Discussion
In this study, we ﬁrst synthesized a disialylated O-linked
glycopeptide, and we then determined the crystal structure of acomplex of LpMab-3-Fab and the glycopeptide. To our knowledge,
this is the ﬁrst 3D structure of a complex between an antibody anda disialyl-core-1 linked glycopeptide. The structure of LpMab-3
reveals that ﬁve of six complementarity-determining regions(CDRs) are used to bind the glycopeptide. The peptide region of theglycopeptide interacts with CDRs of the heavy chain, and the glycan
region is primarily associated with the CDRs of the light chain.
Large antigens such as glycopeptides are difﬁcult to recognize as an
epitope using single-domain antibodies; however, this is possibleusing IgG-type antibodies. This property of IgG provides the potential to generate high speciﬁc mAbs against various types ofglycopeptides.
Importantly, this antibody directly binds to two sialic acid residues of the disialyl-core-1 structure. It is generally established thatthe sialic acid residues of the glycans are involved in facilitatinginﬂuenza virus infections [44,45], the toxicity of cobra toxin [46] orbotulinum toxin [47], the parasitism of malarial parasites [48], andin other processes. This structural information that allows for therecognition of sialic acid by antibodies may be useful for designingneutralizing antibodies that function to bind sialic acid to provide
protection from these biological hazards.

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Author contributions
S.O., Y.K. and T.M. conceived and designed the study. S.O. and
M.K.K. prepared protein sample. M.K.K. sequenced the gene ofantibody. N.K., J.S. and N.T. synthesized the glycopeptide. S.O. and
S.N. analyzed the binding afﬁnity. S.O. and K.S. performed the
crystallization. K.S., S.N. and T.M. processed and reﬁned x-ray data.
S.O., N.K., Y.K. and T.M. wrote the paper. All authors read andapproved the manuscript.
Accession codes
Coordinates and structure factors have been deposited in theProtein Data Bank under accession codes 7C94 (LpMab-3-Fab with
PDPN-glycopeptide) and 7C95 (LpMab-3-Fab without PDPNglycopeptide).
Funding
This research was supported in part by Japan Agency for Medical
Research and Development (AMED) under Grant nos.
JP20am0101083 (T.M.), JP20am0101078 (Y$K.), JP20am0401013(Y$K.) and JP20ae0101028 (Y$K.), and by Grant-in-Aid for ScientiﬁcResearch from Japan Society for the Promotion of Science (JSPS)
under Grant nos. 18H05425 (T.M.), 17K07299 (M.K$K.), and19K07705 (Y$K.).
Declaration of competing interest
The authors declare that they have no known competing
ﬁnancial interests or personal relationships that could haveappeared to inﬂuence the work reported in this paper.
Acknowledgments
The synchrotron radiation experiments were performed at
Photon Factory (proposals 2018G120 and 2018RP-16). We thank thebeamline staff at BL1A of Photon Factory (Tsukuba, Japan) for help
during data collection.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.bbrc.2020.08.103.
References[1] S. Breiteneder-Geleff, K. Matsui, A. Soleiman, P. Meraner, H. Poczewski, R. Kalt,
G. Schaffner, D. Kerjaschki, Podoplanin, novel 43-kd membrane protein ofglomerular epithelial cells, is down-regulated in puromycin nephrosis, Am. J.
Pathol. 151 (1997) 1141e1152.
[2] V. Kasinath, O.A. Yilmam, M. Uehara, M. Yonar, L. Jiang, X. Li, W. Qiu,
S. Eskandari, T. Ichimura, R. Abdi, Urine podoplanin heralds the onset ofischemia-reperfusion injury of the kidney, Am. J. Physiol. Ren. Physiol. 316(2019) F957eF965.
[3] S. Breiteneder-Geleff, A. Soleiman, H. Kowalski, R. Horvat, G. Amann,
E. Kriehuber, K. Diem, W. Weninger, E. Tschachler, K. Alitalo, et al., Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic
capillaries: podoplanin as a speciﬁc marker for lymphatic endothelium, Am. J.
Pathol. 154 (1999) 385e394.
[4] V. Schacht, S.S. Dadras, L.A. Johnson, D.G. Jackson, Y.K. Hong, M. Detmar, Upregulation of the lymphatic marker podoplanin, a mucin-type transmembrane
glycoprotein, in human squamous cell carcinomas and germ cell tumors, Am.
J. Pathol. 166 (2005) 913e921.
[5] E. Jung, D. Gardner, D. Choi, E. Park, Y. Jin Seong, S. Yang, J. Castorena-Gonzalez, A. Louveau, Z. Zhou, G.K. Lee, et al., Development and characterization of
A novel prox1-EGFP lymphatic and schlemm’s canal reporter rat, Sci. Rep. 7(2017) 5577.
[6] K. Suzuki-Inoue, Y. Kato, O. Inoue, M.K. Kaneko, K. Mishima, Y. Yatomi,
Y. Yamazaki, H. Narimatsu, Y. Ozaki, Involvement of the snake toxin receptor

CLEC-2, in podoplanin-mediated platelet activation, by cancer cells, J. Biol.
Chem. 282 (2007) 25993e26001.
[7] Y. Kato, M.K. Kaneko, A. Kunita, H. Ito, A. Kameyama, S. Ogasawara,
N. Matsuura, Y. Hasegawa, K. Suzuki-Inoue, O. Inoue, et al., Molecular analysisof the pathophysiological binding of the platelet aggregation-inducing factor
podoplanin to the C-type lectin-like receptor CLEC-2, Canc. Sci. 99 (2008)
54e61.
[8] Y. Kato, N. Fujita, A. Kunita, S. Sato, M. Kaneko, M. Osawa, T. Tsuruo, Molecular
identiﬁcation of Aggrus/T1alpha as a platelet aggregation-inducing factor
expressed in colorectal tumors, J. Biol. Chem. 278 (2003) 51599e51605.
[9] C.C. Bertozzi, A.A. Schmaier, P. Mericko, P.R. Hess, Z. Zou, M. Chen, C.Y. Chen,
B. Xu, M.M. Lu, D. Zhou, et al., Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling, Blood 116 (2010) 661e670.
[10] A.K. Rishi, M. Joyce-Brady, J. Fisher, L.G. Dobbs, J. Floros, J. VanderSpek,
J.S. Brody, M.C. Williams, Cloning, characterization, and development
expression of a rat lung alveolar type I cell gene in embryonic endodermal andneural derivatives, Dev. Biol. 167 (1995) 294e306.
[11] R. Gonzalez, D. Leaffer, C. Chapin, A.M. Gillespie, W. Eckalbar, L. Dobbs, Cell
fate analysis in fetal mouse lung reveals distinct pathways for TI and TII cell
development, Am. J. Physiol. Lung Cell Mol. Physiol. 317 (2019) L653eL666.
[12] Y. Kato, I. Sasagawa, M. Kaneko, M. Osawa, N. Fujita, T. Tsuruo, Aggrus: adiagnostic marker that distinguishes seminoma from embryonal carcinoma intesticular germ cell tumors, Oncogene 23 (2004) 8552e8556.
[13] E. Martin-Villar, F.G. Scholl, C. Gamallo, M.M. Yurrita, M. Munoz-Guerra,
J. Cruces, M. Quintanilla, Characterization of human PA2.26 antigen (T1alpha2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas, Int. J. Canc. 113 (2005) 899e910.
[14] Y. Kato, M. Kaneko, M. Sata, N. Fujita, T. Tsuruo, M. Osawa, Enhanced
expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing
factor in lung squamous cell carcinoma, Tumor Biol. 26 (2005) 195e200.
[15] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa,J. Hirabayashi, H. Narimatsu, K. Mishima, et al., Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain, Biochem. Biophys. Res.
Commun. 349 (2006) 1301e1307.
[16] K. Mishima, Y. Kato, M.K. Kaneko, Y. Nakazawa, A. Kunita, N. Fujita, T. Tsuruo,
R. Nishikawa, T. Hirose, M. Matsutani, Podoplanin expression in primarycentral nervous system germ cell tumors: a useful histological marker for the
diagnosis of germinoma, Acta Neuropathol. 111 (2006) 563e568.
[17] K. Mishima, Y. Kato, M.K. Kaneko, R. Nishikawa, T. Hirose, M. Matsutani,
Increased expression of podoplanin in malignant astrocytic tumors as a novel
molecular marker of malignant progression, Acta Neuropathol. 111 (2006)
483e488.
[18] S. Abe, Y. Morita, M.K. Kaneko, M. Hanibuchi, Y. Tsujimoto, H. Goto,
S. Kakiuchi, Y. Aono, J. Huang, S. Sato, et al., A novel targeting therapy ofmalignant mesothelioma using anti-podoplanin antibody, J. Immunol. 190(2013) 6239e6249.
[19] A. Kunita, T.G. Kashima, A. Ohazama, A.E. Grigoriadis, M. Fukayama, Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell
migration in osteosarcoma, Am. J. Pathol. 179 (2011) 1041e1049.
[20] Y. Kato, G. Vaidyanathan, M.K. Kaneko, K. Mishima, N. Srivastava,
V. Chandramohan, C. Pegram, S.T. Keir, C.T. Kuan, D.D. Bigner, et al., Evaluationof anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant
gliomas, Nucl. Med. Biol. 37 (2010) 785e794.
[21] A. Kunita, T.G. Kashima, Y. Morishita, M. Fukayama, Y. Kato, T. Tsuruo,
N. Fujita, The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary metastasis, Am. J. Pathol. 170 (2007) 1337e1347.
[22] P. Yuan, S. Temam, A. El-Naggar, X. Zhou, D. Liu, J. Lee, L. Mao, Overexpressionof podoplanin in oral cancer and its association with poor clinical outcome,Cancer 107 (2006) 563e569.
[23] N. Atsumi, G. Ishii, M. Kojima, M. Sanada, S. Fujii, A. Ochiai, Podoplanin, anovel marker of tumor-initiating cells in human squamous cell carcinoma
A431, Biochem. Biophys. Res. Commun. 373 (2008) 36e41.
[24] M. Toyoshima, M. Nakajima, T. Yamori, T. Tsuruo, Puriﬁcation and characterization of the platelet-aggregating sialoglycoprotein gp44 expressed byhighly metastatic variant cells of mouse colon adenocarcinoma 26, Canc. Res.
55 (1995) 767e773.
[25] M. Kaneko, Y. Kato, A. Kunita, N. Fujita, T. Tsuruo, M. Osawa, Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/podoplanin)molecules expressed in Chinese Hamster Ovary cells, J. Biol. Chem. 279 (2004)
38838e38843.
[26] M.K. Kaneko, Y. Kato, A. Kameyama, H. Ito, A. Kuno, J. Hirabayashi, T. Kubota,
K. Amano, Y. Chiba, Y. Hasegawa, et al., Functional glycosylation of human
podoplanin: glycan structure of platelet aggregation-inducing factor, FEBS
Lett. 581 (2007) 331e336.
[27] M. Nagae, K. Morita-Matsumoto, M. Kato, M.K. Kaneko, Y. Kato, Y. Yamaguchi,
A Platform of C-Type lectin-like receptor CLEC-2 for binding O-glycosylated
podoplanin and nonglycosylated rhodocytin, Structure 22 (2014) 1711e1721.
[28] M.K. Kaneko, Y. Kato, T. Kitano, M. Osawa, Conservation of a platelet activating
domain of Aggrus/podoplanin as a platelet aggregation-inducing factor, Gene
378 (2006) 52e57.
[29] T. Sekiguchi, A. Takemoto, S. Takagi, K. Takatori, S. Sato, M. Takami, N. Fujita,
Targeting a novel domain in podoplanin for inhibiting plateletmediated tumor
metastasis, Oncotarget 7 (2015) 3934e3946.
[30] Y. Sayama, M. Sano, T. Asano, Y. Furusawa, J. Takei, T. Nakamura, M. Yanaka,

S. Ogasawara et al. / Biochemical and Biophysical Research Communications 533 (2020) 57e63
S. Okamoto, S. Handa, Y. Komatsu, Y. Nakamura, M. Yanagawa, M.K. Kaneko,
Y. Kato, Epitope mapping of PMab-241, a lymphatic endothelial cell-speciﬁc
anti-bear podoplanin monoclonal antibody, Monoclon. Antibodies Immunodiagn. Immunother. 39 (2020) 77e81.
[31] Y. Kato, M.K. Kaneko, A cancer-speciﬁc monoclonal antibody recognizes theaberrantly glycosylated podoplanin, Sci. Rep. 4 (2014) 5924.
[32] Y. Kato, S. Ogasawara, H. Oki, P. Goichberg, R. Honma, Y. Fujii, M.K. Kaneko,
LpMab-12 established by CasMab technology speciﬁcally detects sialylated Oglycan on Thr52 of platelet aggregation-stimulating domain of human
podoplanin, PLoS One 11 (2016), e0152912.
[33] M.K. Kaneko, H. Oki, Y. Hozumi, X. Liu, S. Ogasawara, M. Takagi, K. Goto,
Y. Kato, Monoclonal antibody LpMab-9 recognizes O-glycosylated N-terminusof human podoplanin, Monoclon. Antibodies Immunodiagn. Immunother. 34(2015) 310e317.
[34] S. Ogasawara, M.K. Kaneko, Y. Kato, LpMab-19 recognizes sialylated O-glycan
on Thr76 of human podoplanin, Monoclon. Antibodies Immunodiagn.
Immunother. 35 (2016) 245e253.
[35] H. Oki, S. Ogasawara, M.K. Kaneko, M. Takagi, M. Yamauchi, Y. Kato, Characterization of monoclonal antibody LpMab-3 recognizing sialylated glycopeptide of podoplanin, Monoclon. Antibodies Immunodiagn. Immunother. 34(2015) 44e50.
[36] M.K. Kaneko, T. Nakamura, R. Honma, S. Ogasawara, Y. Fujii, S. Abe, M. Takagi,
H. Harada, H. Suzuki, Y. Nishioka, et al., Development and characterization ofanti-glycopeptide monoclonal antibodies against human podoplanin, using
glycan-deﬁcient cell lines generated by CRISPR/Cas9 and TALEN, Cancer Med 6(2017) 382e396.
[37] S. Maruyama, K. Suzuki, M. Imamura, H. Sasaki, H. Matsunami, K. Mizutani,
Y. Saito, F.L. Imai, Y. Ishizuka-Katsura, T. Kimura-Someya, M. Shirouzu,
T. Uchihashi, T. Ando, I. Yamato, T. Murata, Metastable asymmetrical structureof a shaftless V1 motor, Sci. Adv. 5 (2019), eaau8149.
[38] W. Kabsch, XDS, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 125e132.
[39] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni,
R.J. Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007)

63658e674.
[40] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta
Crystallogr. Sect. D Biol. Crystallogr. 60 (2004) 2126e2132.
[41] P.D. Adams, P.V. Afonine, G. Bunkoczi, V.B. Chen, I.W. Davis, N. Echols,
J.J. Headd, L.-W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy,
N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson,
T.C. Terwilliger, P.H. Zwart, PHENIX: a comprehensive Python-based system
for macromolecular structure solution, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 213e221.
[42] S.C. Lovell, I.W. Davis, W.B. Arendall III., P.I.W. de Bakker, J.M. Word,
M.G. Prisant, J.S. Richardson, D.C. Richardson, Structure validation by Ca geometry: 4,j and Cb deviation, Proteins Struct. Funct. Genet. 50 (2003)
437e450.
[43] R.A. Laskowski, M.B. Swindells, LigPlotþ: multiple ligand-protein interaction
diagrams for drug discovery, J. Chem. Inf. Model. 51 (2011) 2778e2786.
[44] Y. Suzuki, T. Nakao, T. Ito, N. Watanabe, Y. Toda, G. Xu, T. Suzuki, T. Kobayashi,
Y. Kimura, A. Yamada, et al., Structural determination of gangliosides that bindto inﬂuenza A, B, and C viruses by an improved binding assay: strain-speciﬁc
receptor epitopes in sialo-sugar chains, Virology 189 (1992) 121e131.
[45] R. Wagner, M. Matrosovich, H.D. Klenk, Functional balance between haemagglutinin and neuraminidase in inﬂuenza virus infections, Rev. Med. Virol.
12 (2002) 159e166.
[46] D.C. Gowda, E.A. Davidson, Structural features of carbohydrate moieties insnake venom glycoproteins, Biochem. Biophys. Res. Commun. 182 (1992)
294e301.
[47] K. Niwa, T. Yoneyama, H. Ito, M. Taira, T. Chikai, H. Kouguchi, T. Suzuki,
K. Hasegawa, K. Miyata, K. Inui, T. Ikeda, T. Watanabe, T. Ohyama, Sialic aciddependent binding and transcytosis of serotype D botulinum neurotoxin andtoxin complex in rat intestinal epithelial cells, Vet. Microbiol. 141 (2010)
312e320.
[48] V.K. Goel, X. Li, H. Chen, S.C. Liu, A.H. Chishti, S.S. Oh, Band 3 is a host receptor
binding merozoite surface protein 1 during the Plasmodium falciparum invasion of erythrocytes, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 5164e5169.