Differential Distribution of Podocan Isoforms

Podocan is a relatively novel member of the small leucine‐rich repeat family of proteins. First isolated from podocytes of HIV‐1‐infected kidney tissue, this protein seems to be involved in a number of biological pathways. Initial experiments in podocan‐knockout mice revealed increases in bone density and in vascular smooth muscle cell proliferation in response to injury when compared to the phenotypes of genetically unaltered mice. In addition, protein labeling of human kidney biopsies and vascular lesions has shown both intra and extracellular distribution of podocan. To determine if multiple isoforms of the protein exist, we began a bioinformatics study to align all reported isoforms of human podocan. We found that the previously reported 613 amino acid isoform had a strong N‐terminal signal peptide, as expected for a secreted protein. However, three other isoforms had additional amino acids in front of the signal peptide. We hypothesized that additional sequences upstream of the signal peptide would lead to retention of podocan in the cytoplasm. To test this hypothesis, we fused EGFP to the N‐ or C‐ terminus of secreted podocan. These constructs were subsequently transfected into renal 293T cells. We examined the cells by fluorescent microscopy and western blotting 48 hours later. As predicted, N‐terminal GFP‐podocan prevented efficient secretion of podocan. Our studies have led us to posit that podocan is involved in differential signaling, and this area will be the focus of future investigation. We have generated 3 C‐terminal His‐tagged podocan isoforms that have differing lengths of N‐terminal sequences upstream of the signal peptide and will use these to confirm our GFP‐fusion findings. We have also developed a series of isoform‐specific primers to help us determine whether these isoforms are differentially expressed in normal or diseased human tissue. Grant Funding Source American Association of Anatomists