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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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Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12 and LpMab-7.Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab-7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars: 100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.
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pone.0152912.g002: Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12 and LpMab-7.Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab-7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars: 100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.

Mentions: To confirm the utility of LpMab-12 for immunolabeling of tissues, we compared the reactivity of LpMab-12 with LpMab-7, the most sensitive anti-hPDPN mAb for this type of analysis [29]. Both LpMab-12 (Fig 2A and 2B) and LpMab-7 (Fig 2E and 2F) strongly stained tumor cells in a membranous/cytoplasmic-staining pattern. Lymphatic vessels were immunolabeled clearly without background by LpMab-12 (Fig 2C and 2D) and LpMab-7 (Fig 2G and 2H). Blood vessels were not stained by both mAbs (Fig 2C, 2D, 2G and 2H).


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)

Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12 and LpMab-7.Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab-7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars: 100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152912.g002: Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12 and LpMab-7.Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab-7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars: 100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.
Mentions: To confirm the utility of LpMab-12 for immunolabeling of tissues, we compared the reactivity of LpMab-12 with LpMab-7, the most sensitive anti-hPDPN mAb for this type of analysis [29]. Both LpMab-12 (Fig 2A and 2B) and LpMab-7 (Fig 2E and 2F) strongly stained tumor cells in a membranous/cytoplasmic-staining pattern. Lymphatic vessels were immunolabeled clearly without background by LpMab-12 (Fig 2C and 2D) and LpMab-7 (Fig 2G and 2H). Blood vessels were not stained by both mAbs (Fig 2C, 2D, 2G and 2H).

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