Podocalyxin in Rat Platelets and Megakaryocytes

A Miettinen

M Solin

J Reivinen

E Juvonen

R Väisänen

H Holthöfer

From the Division of Bacteriology and Immunology,* Haartman Institute, University of Helsinki, and the HUCH Diagnostics† and Department of Medicine‡ , University Central Hospital, Helsinki, Finland

Accepted 1998 Nov 30.

Abstract

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent M_r of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

Abstract

Podocalyxin was first described as an integral membrane protein covering the luminal membranes of glomerular podocytes and endothelial cells in the rat.^1-4 Thus far it has not been described in other cell types. It is highly glycosylated with N- and O-linked carbohydrates, both of which are sialylated and sulfated.⁵ Its localization at the apical membranes and its strong anionic charge suggest that it may help to maintain open vascular lumens and functional glomerular filtration slits.^5-7 Podocalyxin-like proteins (PCLPs) have been cloned from chicken,⁸ rabbit,⁹ and man.¹⁰ In all of these species the intracellular and transmembrane domains of the proteins are highly homologous. The extracellular parts are more heterogeneous, but all share a mucin-like structure and four conserved cysteins. Tissue distribution, biochemical characteristics, and genomic features¹⁰ of these proteins resemble each other, but no sequence data of the rat protein have been published so far. The chicken PCLP thrombomucin is present in myeloid stem cells, megakaryocytes, and thrombocytes⁸ and structurally resembles CD34 antigen, but the mammalian PCLPs have not been described in hematopoietic cells.

We have earlier characterized monoclonal antibodies specific for rat podocalyxin.^3,4 In preliminary experiments we noticed that after intravenous injections into rats the antibodies bound to endothelial cells³ and sometimes were detected at the surface of platelets in glomeruli (A. Miettinen, unpublished observations). This prompted us to study whether podocalyxin is present in platelets. Here we show, using biochemical, immunological, and histological techniques, that podocalyxin is expressed in rat platelets and megakaryocytes. We have also partially cloned rat glomerular podocalyxin, present the first sequence data of it, and demonstrate using molecular biology techniques that podocalyxin mRNA is present in megakaryocytes and platelets.

Acknowledgments

We thank Dr. David B. Kershaw for generously providing us with the rabbit PCLP1 cDNA and Dr. E. Chignier for LYP-20 antibodies. Leiras Pharmaceutical Co., Helsinki, Finland, is acknowledged for giving us Ilomedin.

Acknowledgments

Footnotes

Address reprint requests to: Dr. Aaro Miettinen, Division of Bacteriology and Immunology, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. E-mail: .if.iknisleh@nenitteim.oraa

Supported by grants from Helsinki University Central Hospital, The Sirkka and Sakari Sohlberg Foundation, The Paulo Foundation, and The Academy of Finland.

Footnotes

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