Heterologer expression der L-aminosauren Oxydase vom calloselasma rhodostoma und Induction der apoptose

It has been proposed that L-amino acid oxidases (LAAOs) from different snake venoms interact with the cell membrane receptors to induce many of their toxic effect. This interaction could be mediated by the terminal sialic acids of the N-linked glycan moieties. However no clear and direct evidence has been proposed so far. One of the glycan moieties of LAAO from Calloselasma rhodostoma (CRLAAO) is located near the vicinity of the substrate entry channel, which also serves for the release of the products including the cytotoxic H2O2. To verify the above-mentioned hypothesis, one should have to compare the modes of action by glycosylated and unglycosylted LAAOs. Attempts were made to obtain unglycosylated LAAO either by enzymatic (PNGase-F) treatment or by heterologous expression in E.coli. But no soluble active LAAO could be obtained in either case. Moreover expression in Baculovirus and mammalian expression systems led to secretion of either very little and (or) inactive recombinant enzyme. Expression in Pichia pastoris was successful to some extent in that the active recombinant LAAO could be expressed and purified to near homogeneity. Further standardizations are required inorder to improve the expression yields for characterization of the recombinant enzyme. As an alternative method desialylated native LAAO was also prepared by treatment with neuraminidase and this enzyme was used in the cell culture studies. It was shown that sialic acids have a role in binding of LAAO to the cell surface and internalization, which might be a mechanism to potentiate the effects of H2O2 by enhancing its local concentration. Once being internalized LAAO might further exert toxic effects by generating hydrogen peroxide and metabolizing amino acids and/or other factors important for cell survival. The possible sialic acid receptors mediating the binding of this toxic LAAO are yet to be identified.