Podocalyxin in rat platelets and megakaryocytes

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 Mr 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.

Figure 1.

Podocalyxin antigens in rat kidney (a) and platelets (b). Rabbit anti-podocalyxin antibodies (290) give a typical staining of glomerular podocytes and peritubular endothelial cells, as shown by indirect IF staining at cryostat sections of rat kidney (a). Smear slides of rat peripheral blood stained with monoclonal 5A anti-podocalyxin (b) or anti-CD62P (c) antibodies. The antibodies bind to platelets but not to red blood cells or leukocytes (arrows). Note the granular staining pattern of acetone-fixed platelets. Bar, 100 μm (a), 10 μm (b and c).

Figure 2.

Podocalyxin (a) and CD62P antigens (b) in a megakaryocyte, shown by double IF staining of a cytospin slide of rat bone marrow cells first with monoclonal anti-CD62P and TRITC-conjugated goat anti-mouse IgG (b), followed by FITC-conjugated 5A IgG (a). Binding to the anti-mouse conjugate was blocked by pretreatment with normal mouse serum. The polyploid megakaryocyte expresses both antigens. Anti-podocalyxin antibodies detect also a small, unidentified cell (a, arrow). Magnification, ×600.

Figure 3.

Podocalyxin proteins of rat platelets and glomeruli. A: SDS-PAGE analysis of a Triton X-100 extract from isolated rat glomeruli (lane 1) and of the purified podocalyxin used for amino acid analysis or immunizations (silver staining). B and C: Western blotting of extracts of freshly isolated platelets (lane 1) or of rat glomeruli (lane 2). with rabbit (290, B) or mouse monoclonal (5A, C) anti-podocalyxin antibodies. Note slower mobility of platelet podocalyxin as compared with the podocalyxin forms of the glomerular extract.

Figure 4.

Ultrastructural localization of podocalyxin in freshly isolated platelets. Colloidal gold particles indicating the presence of podocalyxin are detected at intracellular membranes in all platelets after staining with monoclonal anti-podocalyxin (a, arrows) but not with the control anti-megalin (b). IgG. At higher magnification, gold particles are seen in an α-granule (c, arrows) and at the surface of a platelet. Post-embedding indirect immunogold staining; magnification, ×10,000 (a and b) and ×36,000 (c).

Figure 5.

Surface expression of podocalyxin or CD62P antigens at freshly isolated (left panel) or thrombin-treated (right panel), nonpermeabilized platelets. Both anti-podocalyxin and anti-CD62P IgGs bind to some extent to the surface of isolated platelets, indicating slight activation of the cells. A significant increase in the expression of podocalyxin or CD62P is seen after thrombin stimulation. The x axis shows fluorescence intensity.

Figure 6.

Amino acid sequence alignment of rat podocalyxin (derived from clone 51A) and the previously published podocalyxin-like proteins of rabbit, human, and chicken (gallus). Note the identical or highly homologous structure of the putative transmembrane (double underlining) and intracellular domains of all species. The intracellular domain of rat podocalyxin shows one protein kinase C(*) and two casein kinase II (○) consensus phosphorylation sites, and its partially cloned extracellular domain extends to include two of the four conserved cysteins (arrows). The extracellular seven-amino-acid sequence (aa 50 to 56, line above) was found independently by amino acid sequencing of tryptic peptides of isolated rat glomerular podocalyxin. The probes used for Northern blotting or in situ hybridization covered the whole area shown. Numbering is according to the sequences in GenBank. Accession numbers are U35239 rabbit PCPL-1, U97519 human PCLP, Y13978 Gallus gallus thrombomucin.

Figure 7.

Northern blot of mRNA extracted from rat renal cortex (left lane) or cultured rat renal epithelial NRK cells (right lane) developed using the 589-bp rat-podocalyxin probe. The probe binds to a 5.5-kb transcript of renal cortical but not NRK mRNA. The lower arrowhead shows the position of the 1.9-kb β-actin mRNA. The size of the podocalyxin transcript is deduced from results obtained with renal cortical total RNA (not shown).

Figure 8.

Micrographs demonstrating in situ hybridization of antisense (a and b) or sense (c and d) cRNA probes of rat podocalyxin to rat kidney sections. The antisense probe decorates glomerular (G) podocytes and all endothelial cells (arrow shows an arteriole), whereas the sense probes do not show any specific binding. a and c, direct light; b and d, phase contrast. Magnification, ×200.

Figure 9.

Micrographs demonstrating in situ hybridization of antisense (a and b) cRNA probes of rat podocalyxin to large megakaryocytic cells (arrows) at cytospin preparations of rat bone marrow. No binding over the relatively high background is seen with the sense probes (c and d). a and c, dark field; b and d, respective transmission light micrographs. Hematoxylin counterstaining; magnification, ×630.