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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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Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry.(A) CHO-K1 cells were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus (WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used as well to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used as internal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B) CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).
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pone.0152912.g003: Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry.(A) CHO-K1 cells were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus (WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used as well to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used as internal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B) CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).

Mentions: To determine the critical epitope for the LpMab-12 interaction with hPDPN, we compared the mAb binding to the hPDPN carrying different point mutations. Using Western blot, we found that LpMab-12 did not detect protein sequences with the following amino acid substitutions: D49A, V51A, T52A, and P53A (Fig 3A).


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)

Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry.(A) CHO-K1 cells were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus (WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used as well to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used as internal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B) CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816300&req=5

pone.0152912.g003: Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry.(A) CHO-K1 cells were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus (WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used as well to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used as internal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B) CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).
Mentions: To determine the critical epitope for the LpMab-12 interaction with hPDPN, we compared the mAb binding to the hPDPN carrying different point mutations. Using Western blot, we found that LpMab-12 did not detect protein sequences with the following amino acid substitutions: D49A, V51A, T52A, and P53A (Fig 3A).

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