2. CHAPTER: I
4.6 C ONCLUSION
LAAO, a major component of snake venom, is a potent cytotoxin acting primarily via the production of high amounts of H2O2, which kill the cells by necrosis. However, LAAO retains its cytotoxicity also in the presence of antioxidants. In this case the mode of cell demise switches to apoptosis. Apoptosis induction by LAAO involves at least two different mechanisms, the first one being an alteration of the composition of the growth medium, in particular of its serum components. Secondly, immunoblot experiments show that LAAO is retained by Jurkat cells where it undergoes specific proteolysis. This is suggestive of an interaction of the enzyme with the cell surface, a process which seems to require its glycan moieties. The study of the mechanisms of action of animal toxins is an important step toward their potential application as anticancer agents.
Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft (DFG-grants Gh 2/12-1 to S. G. and MA 2385/2-2 to E. F.-M.)
We thank Eva-Maria Boneberg for help in performing FACS analysis and Alexander Bürkle for critical reading of the manuscript.
5 SUMMARY
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 effects [21], [49]. 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.[17].
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.
6 ZUSAMMENFASSUNG
Es wird angenommen, dass L-Aminosäure-Oxidasen (LAAOs) unterschiedlicher Schlangengifte mit Rezeptoren in Zellmembranen (bzw.
Zellmembranrezeptoren) interagieren, um viele ihrer toxischen Effekte hervorzurufen. Diese Interaktionen könnten durch N-verbrückte Glycanbestandteile terminaler Sialinsäuren vermittelt werden. Hierfür konnte jedoch noch kein klarer und direkter Beweis geliefert werden. Einer dieser Glycanbestandteile von LAAO aus Calloselasma rhodostoma (CRLAAO) liegt in Nachbarschaft des Substrat-Eingangskanals, welcher ebenso zur Freisetzung von Stoffwechselprodukten einschliesslich zellschädigendem H2O2 dient. Um diese Hypothese zu verifizieren müsste man die Wirkungsweise (“mode of action”) glycosylierter und unglycosylierter LAAOs vergleichen. Es wurden Versuche unternommen, unglycosylierte LAAO entweder durch enzymatische Behandlung (PNGase-F) oder heterologe Expression in E. coli zu erhalten. Jedoch konnte in keinem der beiden Fälle lösliche aktive LAAO erhalten werden. Darüber hinaus führte Expression in Baculoviren und Säugetierzellen entweder zur Sekretion von sehr wenig und/oder inaktivem rekombinantem Enzym. Expression in Pichia pastoris war insofern erfolgreich, als dass aktive rekombinante LAAO exprimiert und nahezu bis zu vollständiger Homogenität gereinigt werden konnte. Weitere Optimieungen sind notwendig, um die Expressions-Ausbeute zur Charakterisierung des rekombinanten Enzyms zu erhöhen. Als alternative Methode wurde de-sialysierte, native LAAO durch Behandlung mit Neuraminidase hergestellt und in Zellkultur-Studien verwendet. Es wurde gezeigt, dass Sialinsäuren bei der Bindung von LAAO an die Zelloberfläche und bei deren Internalisierung eine Rolle spielen. Letzteres könnte ein Mechanismus sein, um die Effekte von H2O2 durch lokale Konzentrationserhöhung zu verstärken. Ist LAAO erst einmal internalisiert, so könnte diese weitere toxische Effekte durch Herstellung von Wasserstoffperoxid und Metabolisierung von Aminosäuren und/oder anderer zum Überleben der Zellen wichtiger Faktoren ausüben. Mögliche, die Bindung dieser toxischen LAAO vermittelnde, Sialinsäure-Rezeptoren sind noch nicht identifiziert.
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