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Cell biology of Dpl in cultured cells

Like PrP, Dpl is shed from the surface of cells through the action of a zinc metalloprotease. Whilst the majority of PrPC is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. PrPC can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrPC was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the secretase-mediated shedding of the amyloid precursor protein (APP) indicating a proteolytic shedding mechanism.

Like APP, this zinc metalloprotease-mediated shedding of PrPC could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft disrupting agents filipin and methylbeta-cyclodextrin promoted the shedding of PrPC via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrPC is likely to occur via phospholipase cleavage of the GPI anchor as a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, these data indicate that shedding of PrPC can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.

Unlike PrP, Dpl is not endocytosed upon exposure of cells to copper or zinc. In collaboration with the laboratory of Pr Laplanche, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrPc on mature spermatozoa. The localisation of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility.

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