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      PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin

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          Abstract

          Sensitive and specific monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb, PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)–K1 (CHO/gPDPN) cells, and screened mAbs against gPDPN using flow cytometry. One of the mAbs, PMab-235 (IgG 1, kappa), specifically detected CHO/gPDPN cells by flow cytometry. Furthermore, PMab-235 strongly detected lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. These findings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

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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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              Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.

              Naoya Fujita, Shigeo Sato, Yukinari Kato … (2003)
              Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.
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                Author and article information

                Contributors
                Yukinari Kato
                Journal
                Journal ID (nlm-ta): Heliyon
                Journal ID (iso-abbrev): Heliyon
                Title: Heliyon
                Publisher: Elsevier
                ISSN (Electronic): 2405-8440
                Publication date PMC-release: 10 July 2019
                Publication date Collection: July 2019
                Publication date (Electronic): 10 July 2019
                Volume: 5
                Issue: 7
                Electronic Location Identifier: e02063
                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
                [c ]ZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima, 963-0196, Japan
                [d ]Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
                Author notes
                Article
                Publisher ID: S2405-8440(19)35723-8 Publisher ID: e02063
                DOI: 10.1016/j.heliyon.2019.e02063
                PMC ID: 6626078
                SO-VID: 7f934f89-2ab6-404d-af7b-93233d33fcfe
                Copyright © © 2019 The Author(s)
                License:

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

                History
                Date received : 18 February 2019
                Date revision received : 10 May 2019
                Date accepted : 5 July 2019
                Categories
                Subject: Article

                Keywords: lymphatic endothelial cells,pmab-235,goat podoplanin,pdpn
                Data availability:
                Keywords: lymphatic endothelial cells, pmab-235, goat podoplanin, pdpn

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