Development of Monoclonal Antibody LpMab-10 Recognizing Non-glycosylated PLAG1/2 Domain Including Thr34 of Human Podoplanin.
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.
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .
Satoshi
S
Ogasawara
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .|2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Hiroharu
H
Oki
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .
Mika K
MK
Kaneko
3 Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine , Yamagata, Japan .
Yasukazu
Y
Hozumi
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .|2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Xing
X
Liu
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .|2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Ryusuke
R
Honma
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .
Yuki
Y
Fujii
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .
Takuro
T
Nakamura
3 Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine , Yamagata, Japan .
Kaoru
K
Goto
2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Michiaki
M
Takagi
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Miyagi, Japan .
Yukinari
Y
Kato
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D037181:Lectins, C-Type / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology*; D010974:Platelet Aggregation / Q000276:immunology; D011134:Polysaccharides / Q000276:immunology; D017434:Protein Structure, Tertiary; D000098853:Podoplanin
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011134:Polysaccharides; C104778:CLEC2B protein, human; C117468:PDPN protein, human; D000098853:Podoplanin; C104382:PLAG1 protein, human