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Cancer Med. 2017 Feb;6(2):382-396. doi: 10.1002/cam4.954. Epub 2017 Jan 19.

Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

Author information

1
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
2
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata, 990-9585, Japan.
3
Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima, 770-8505, Japan.
4
Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
5
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.
6
Department of Pathology and Laboratory Medicine, Sendai Medical Center, 2-8-8, Miyagino, Miyagino-ku, Sendai, Miyagi, 983-0045, Japan.

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.

KEYWORDS:

Epitope; glycopeptide; monoclonal antibody; podoplanin; sialic acid

PMID:
28101903
PMCID:
PMC5313638
DOI:
10.1002/cam4.954
[Indexed for MEDLINE]
Free PMC Article

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