title: Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.
abstract: Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN.
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors.forename: Mika K
authors.initials: MK
authors.lastname: Kaneko
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.|Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata, 990-9585, Japan.
authors.forename: Ryusuke
authors.initials: R
authors.lastname: Honma
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ogasawara
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fujii
authors.affiliation: Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima, 770-8505, Japan.|Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Abe
authors.affiliation: Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata, 990-9585, Japan.
authors.forename: Michiaki
authors.initials: M
authors.lastname: Takagi
authors.affiliation: Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Harada
authors.affiliation: Department of Pathology and Laboratory Medicine, Sendai Medical Center, 2-8-8, Miyagino, Miyagino-ku, Sendai, Miyagi, 983-0045, Japan.
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Nishioka
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors.forename: Yukinari
authors.initials: Y
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology*; D016466:CHO Cells; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D009928:Organ Specificity; D000098853:Podoplanin
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D008562:Membrane Glycoproteins; C117468:PDPN protein, human; D000098853:Podoplanin
keywords: Epitope; glycopeptide; monoclonal antibody; podoplanin; sialic acid