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      Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

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          Abstract

          Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues because PDPN is known as a lymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil. However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using flow cytometry. One of the mAbs, PMab-247 (IgG 1, kappa), specifically detected CHO/bPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-247 for the functional analyses of bPDPN.

          Highlights

          • •

            PDPN is known as a specific lymphatic endothelial cell (LEC) marker.

          • •

            Sensitive and specific PMab-247 mAb against bear PDPN was produced.

          • •

            PMab-247 strongly reacted with bear PDPN in flow cytometry.

          • •

            PMab-247 is useful for IHC using paraffin-embedded cell sections.

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          Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells.

          Yukinari Kato, Yutaka Yatomi, Katsue Suzuki-Inoue … (2007)
          Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.
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            Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

            Christopher Hess, Thomas Graf, Matthew Santore … (2010)
            Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.
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              Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.

              H Poczewski, S Geleff, K Matsui … (1997)
              Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin.
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                Author and article information

                Contributors
                Yukinari Kato
                Journal
                Journal ID (nlm-ta): Biochem Biophys Rep
                Journal ID (iso-abbrev): Biochem Biophys Rep
                Title: Biochemistry and Biophysics Reports
                Publisher: Elsevier
                ISSN (Electronic): 2405-5808
                Publication date PMC-release: 28 April 2019
                Publication date Collection: July 2019
                Publication date (Electronic): 28 April 2019
                Volume: 18
                Electronic Location Identifier: 100644
                Affiliations
                [a ]Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
                [b ]New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
                Author notes
                [∗ ]Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan, yukinarikato@ 123456med.tohoku.ac.jp
                Article
                Publisher ID: S2405-5808(19)30084-6 Publisher ID: 100644
                DOI: 10.1016/j.bbrep.2019.100644
                PMC ID: 6488525
                SO-VID: 2666a897-8680-43b5-90c6-92929d3bb70f
                Copyright © © 2019 The Authors
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 6 April 2019
                Date revision received : 20 April 2019
                Date accepted : 22 April 2019
                Categories
                Subject: Research Article

                Keywords: bear podoplanin,pdpn,immunohistochemistry,pmab-247,bap, bovine aggrus/podoplanin,dab, 3,3′-diaminobenzidine tetrahydrochloride,elisa, enzyme-linked immunosorbent assay,pbs, phosphate-buffered saline,pdpn, podoplanin,mab, monoclonal antibody
                Data availability:
                Keywords: bear podoplanin, pdpn, immunohistochemistry, pmab-247, bap, bovine aggrus/podoplanin, dab, 3,3′-diaminobenzidine tetrahydrochloride, elisa, enzyme-linked immunosorbent assay, pbs, phosphate-buffered saline, pdpn, podoplanin, mab, monoclonal antibody

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