Fig. 5

The divergent zebrafish Pst polysialyltranferase shows a low affinity for NCAM and autopolysialisation in vitro. (A) Sepharose fixed Protein A-fusion proteins of zebrafish Pst and Stx were analyzed in vitro for NCAM polysialylation. The radiolabelled reaction products were analyzed by SDS-PAGE and autoradiography before (-) and after (+) treatment with endoN. Depolysialylated Protein A fusion proteins of enzymes and NCAM are indicated. Under these conditions only zebrafish Stx is able to polysialylate NCAM to the same amount as the control, hamster Pst. (B) The western blot immunostained with anti-Protein A mouse IgG demonstrates that loading of beads with recombinant protein A-polysialyltransferases was comparable in all reaction mixtures. (C) In vivo activity of zebrafish polysialyltransferases using NCAM- and polysialyltransferase-negative LM-TK cells. Cells were analyzed for PSA expression by immunoflourescence using anti-PSA mAb 735 (red). Nuclei were visualized by Hoechst staining (blue). Cells transfected either with an empty vector or solely with NCAM do not express PSA (mock, mock-NCAM). The expression of one polysialyltransferase (zPST, zSTX), either alone or in co-transfection with NCAM, leads to a strong PSA signal comparable to the positive controls (hPST, mSTX). In cells expressing solely one polysialyltransferase, the golgi apparatus (arrows) is labelled for PSA, whereas co-expression with NCAM leads to an additional labelling of the cell membrane (arrowheads). An exception is cells transfected with zebrafish Pst alone, that reveal expression of PSA in both structures.