Deglycosylated ceruloplasmin maintains its enzymatic, antioxidant, cardioprotective, and neuronoprotective properties

Aouffen, M'hammed; Paquin, Joanne; Grandpré, Eric De; Nadeau, Réginald; Mateescu, Mircea-Alexandru
August 2001
Biochemistry & Cell Biology;Aug2001, Vol. 79 Issue 4, p489
Academic Journal
Ceruloplasmin (CP), an important serum antioxidant, is a blue copper glycoprotein with ferroxidase and oxidase activities. Among other physiological actions, plasma CP was shown to protect isolated rat hearts and cultured P19 neurons exposed to oxidative stress conditions, raising the possibility of using this protein in the treatment of cardiac and neuronal diseases related to oxidative damage. However, since therapeutic applications of CP must be compatible with restrictions in the administration of blood derivatives to humans, there is a need to produce the protein by genetic engineering. To help in the choice of adequate expression systems, we undertook this study to determine if the carbohydrate moiety on the protein is essential for its functions. CP was completely deglycosylated using N-glycosidase F under nondenaturing conditions. Deglycosylated CP was found to retain most of the conformational, antioxidant, and enzymatic properties of the native protein in vitro. Moreover, both forms of the protein had similar cardioprotective and neuronoprotective effects against oxidative stress as evaluated with isolated rat hearts undergoing ischemia–reperfusion and with cultured P19 neurons exposed to xanthine – xanthine oxidase. The data thus indicate that the carbohydrate moiety of CP is not essential for its enzymatic and protective actions. Accordingly, even the use of expression systems that do not glycosylate mammalian proteins could provide a recombinant CP that retains its therapeutic potential.Key words: copperproteins, protein-linked carbohydrates, ischemia-reperfusion, isolated rat hearts, cultured P19 neurons.La céruloplasmine (CP), un important antioxydant du sérum, est une glycoprotéine à cuivre bleue avec des activités ferroxydasique et oxydasique. Parmi d'autres fonctions physiologiques, il a été montré que la CP protège le cœur de rat isolé et les neurones P19 en culture lorsqu'ils sont exposés à des conditions de stress oxydatif, soulevant ainsi la possibilité d'utiliser cette protéine dans le traitement de maladies cardiaques et neuronales liées à des dommages oxydatifs. Cependant, comme les applications thérapeutiques de la CP doivent composer avec certaines restrictions quant à l'utilisation des dérivés du sang pour administration chez l'humain, il y a donc un besoin de produire la protéine par génie génétique. Pour aider à choisir un système d'expression adéquat, nous avons réalisé cette étude pour déterminer si les glucides présents sur la protéine sont essentiels à ses fonctions. La CP a été complètement déglycosylée par traitement avec la N-glycosidase F en conditions non dénaturantes. Il s'est avéré que la CP déglycosylée conserve l'ensemble des propriétés conformationnelles, antioxydantes et enzymatiques de la protéine native. De plus, les deux formes de la CP ont des effets cardioprotecteur et neuronoprotecteur semblables contre le stress oxydatif, tel qu'évalué avec des cœurs de rat isolés soumis à l'ischémie–reperfusion et avec des cultures de neurones P19 exposées au système xanthine – xanthine oxydase. Les données indiquent donc que la partie glucidique de la CP n'est pas essentielle pour ses actions enzymatiques et protectrices. Par conséquent, même l'utilisation de systèmes d'expression qui ne glycosylent pas les protéines de mammifères pourrait fournir une CP recombinante qui conserverait son potentiel thérapeutique.Mots clés : protéines à cuivre, glucides liés à des protéines, ischémie-reperfusion, cœurs de rat isolés, culture de neurones P19.


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