Development of Monoclonal Antibody LpMab-10 Recognizing Non-glycosylated PLAG1/2 Domain Including Thr34 of Human Podoplanin

Satoshi Ogasawara, Hiroharu Oki, Mika K. Kaneko, Yasukazu Hozumi, Xing Liu, Ryusuke Honma, Yuki Fujii, Takuro Nakamura, Kaoru Goto, Michiaki Takagi, Yukinari Kato

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein that possesses a platelet aggregation-stimulating (PLAG) domain in the N-terminus. PLAG domain includes three tandem repeats of eight amino acids: PLAG1, PLAG2, and PLAG3. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. In contrast, the glycosylation of Thr34 of PLAG1 of human PDPN remains to be clarified. Herein, we developed and characterized a novel anti-PDPN monoclonal antibody, LpMab-10, which targets PLAG1/2 domain. LpMab-10 detects endogenous PDPN of cancer cells and normal cells independently of glycosylation. The minimum epitope of LpMab-10 was identified as Glu33-Gly45 of PDPN using Western blot and flow cytometry. The Thr34 of PLAG1 is critical for LpMab-10 recognition, and O-glycan is not included in LpMab-10 epitope, indicating that Thr34 of PLAG1 is not O-glycosylated. In immunocytochemical and immunohistochemical analyses, LpMab-10 strongly detected PDPN-expressing tumor cells. By using monoclonal antibodies against different Ser/Thr, including epitopes of PDPN, it becomes possible to determine whether Ser/Thr residues of PDPN are O-glycosylated.

Original languageEnglish
Pages (from-to)318-326
Number of pages9
JournalMonoclonal antibodies in immunodiagnosis and immunotherapy
Volume34
Issue number5
DOIs
Publication statusPublished - 2015 Oct

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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