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LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin.

Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii Y, Kaneko MK - PLoS ONE (2016)

Bottom Line: The minimal epitope of LpMab-12 was identified as Asp49-Pro53 of hPDPN.Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38-54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2-6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52.LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN.

View Article: PubMed Central - PubMed

Affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

ABSTRACT
Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49-Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38-54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2-6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.

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Schematic summary of the epitopes for several anti-hPDPN mAbs.Glycosylation sites are shown (O-glycan). Numbers indicate amino acid position. GpMab, anti-glycopeptide mAb; PLAG, platelet aggregation-stimulating.
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pone.0152912.g005: Schematic summary of the epitopes for several anti-hPDPN mAbs.Glycosylation sites are shown (O-glycan). Numbers indicate amino acid position. GpMab, anti-glycopeptide mAb; PLAG, platelet aggregation-stimulating.

Mentions: To further clarify the essential epitope of LpMab-12, especially the essential glycan structure detected by LpMab-12, we synthesized several glycopeptides of hPDPN, which include the PLAG2 and PLAG3 domains (Fig 4). Specifically, we generated SAα2-6GalNAc + hpp3854; Gal + GalNAc + hpp3854; SAα2-3Gal + GalNAc + hpp3854; Gal + SAα2-6GalNAc + hpp3854; and SAα2-3Gal + SAα2-6GalNAc + hpp3854. LpMab-12 detected SAα2-6GalNAc + hpp3854, Gal + SAα2-6GalNAc + hpp3854, and SAα2-3Gal + SAα2-6GalNAc + hpp3854 (Table 1). LpMab-9, the epitope of which was identified as residues 25–30 of hPDPN [26], did not react with any glycopeptides of hpp3854. In contrast, LpMab-13 and LpMab-20, which were recently established using CasMab technology [30], recognized all the glycopepties of hpp3854. Collectively, these data indicate that the essential epitope of LpMab-12 is 49-DVVT(SAα2-6GalNAc)P-53 (Fig 5).


LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin.

Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii Y, Kaneko MK - PLoS ONE (2016)

Schematic summary of the epitopes for several anti-hPDPN mAbs.Glycosylation sites are shown (O-glycan). Numbers indicate amino acid position. GpMab, anti-glycopeptide mAb; PLAG, platelet aggregation-stimulating.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816300&req=5

pone.0152912.g005: Schematic summary of the epitopes for several anti-hPDPN mAbs.Glycosylation sites are shown (O-glycan). Numbers indicate amino acid position. GpMab, anti-glycopeptide mAb; PLAG, platelet aggregation-stimulating.
Mentions: To further clarify the essential epitope of LpMab-12, especially the essential glycan structure detected by LpMab-12, we synthesized several glycopeptides of hPDPN, which include the PLAG2 and PLAG3 domains (Fig 4). Specifically, we generated SAα2-6GalNAc + hpp3854; Gal + GalNAc + hpp3854; SAα2-3Gal + GalNAc + hpp3854; Gal + SAα2-6GalNAc + hpp3854; and SAα2-3Gal + SAα2-6GalNAc + hpp3854. LpMab-12 detected SAα2-6GalNAc + hpp3854, Gal + SAα2-6GalNAc + hpp3854, and SAα2-3Gal + SAα2-6GalNAc + hpp3854 (Table 1). LpMab-9, the epitope of which was identified as residues 25–30 of hPDPN [26], did not react with any glycopeptides of hpp3854. In contrast, LpMab-13 and LpMab-20, which were recently established using CasMab technology [30], recognized all the glycopepties of hpp3854. Collectively, these data indicate that the essential epitope of LpMab-12 is 49-DVVT(SAα2-6GalNAc)P-53 (Fig 5).

Bottom Line: The minimal epitope of LpMab-12 was identified as Asp49-Pro53 of hPDPN.Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38-54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2-6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52.LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN.

View Article: PubMed Central - PubMed

Affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

ABSTRACT
Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49-Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38-54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2-6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.

Show MeSH
Related in: MedlinePlus