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[Xanthine Oxidase] in fjU/ml

Fig. 3. N2A neuroblastoma cells were transfected with a construct (pCDNA3) expressing PrPC. Cells expressing large amounts of PrPC (•) are more resistant to the toxicity of superoxide as generated by xanthine oxidase (in the presence of 40 U/mL of catalase) than non transfected cells (O). However, infection of these cells with the ME7 strain of scrapie so that the cells generate PrPSc (■) renders them much more sensitive to superoxide toxicity. Survival was determined using an MTT based assay. Shown are the mean and SEM of four separate experiments.

Analysis of recombinant mouse and chicken prion protein has led to the discovery of an important gain of function once PrPC binds copper (55). Recombinant PrPC with at least two atoms of copper bound specifically via the octameric-repeat region has an activity like that of superoxide dismutase (SOD), implying that the prion protein may act to detoxify superoxide, thus preventing oxidative stress from occurring (67). PrPC binding four atoms of copper has higher activity than that binding only two. However, the activity is enzymatic, because copper forced to bind to a mutant prion protein lacking the octameric region does not endow the protein with this antioxidant activity (55). There is also evidence that SOD by the prion protein leads to specific oxidation of methionines that are clustered in the C-terminal of the protein (68). Although many SOD remove the extra electron of superoxide by forming hydrogen peroxide, it is also possible that the electron can be removed by rapid oxidation of methionines. PrPC is rapidly turned over and part of its metabolic breakdown involves cleavage of the N-terminal at a site that lies between the octameric repeat and the methionine-rich globular C-terminal domain. Thus, separating the copper from what is probably the active site of the protein in its catalytic form.

These results have also been confirmed with native PrPC purified from either the brains of mice or from cultured neurones (56). PrPC purified from mouse brain has three atoms of Cu bound per molecule. This is sufficient to endow it with SOD activity. By growing neurones in culture under conditions of low copper, it is possible for cells to express PrPC, which has very low Cu bound. It was not possible to isolate PrPC from cells grown under low-Cu conditions with less than one atom of Cu bound per molecule. Such purified protein lacked SOD activity. Neurones grown under different copper conditions could be induced to express PrPC with one, two, three, or four atoms of copper bound. Increasing Cu concentration in the cell culture medium to 25 ^M (which is toxic) did not

Fig. 4. Circular dichrosim spectra analysis of recombinant mouse PrP of different alleles. Recombinant protein was produced from E. coli based either on the a allele (PrPa) or the b allele (PrPb) The far UV spectra for PrP samples were analyzed using circular dichroism spectroscopy. Samples were refolded to bind copper and measured immediately or when aged for 4 wk. Shown are fresh PrPa (thin unbroken line), PrPb (thick dotted light) aged PrPa (thick unbroken line) and aged PrPb (thick dotted line). Values are expressed as molar ellipticity (0) for 190-250 nm.

Fig. 4. Circular dichrosim spectra analysis of recombinant mouse PrP of different alleles. Recombinant protein was produced from E. coli based either on the a allele (PrPa) or the b allele (PrPb) The far UV spectra for PrP samples were analyzed using circular dichroism spectroscopy. Samples were refolded to bind copper and measured immediately or when aged for 4 wk. Shown are fresh PrPa (thin unbroken line), PrPb (thick dotted light) aged PrPa (thick unbroken line) and aged PrPb (thick dotted line). Values are expressed as molar ellipticity (0) for 190-250 nm.

Fig. 5. The highest concentration of the prion protein is found at synapses. It has been found that expression of prion protein increases the amount of copper found at and released by synapses. It is possible that the prion protein could be released along with copper bound to it. Alternatively, copper could be captured by PrP on membranes on either side of the synpatic cleft. However, studies on the effects of copper on synapses suggest that the absence of prion protein expression makes synapse more susceptible to the deleterious effects of copper on neurotransmission implying that the prion protein is expressed there to have a protective role.

Fig. 5. The highest concentration of the prion protein is found at synapses. It has been found that expression of prion protein increases the amount of copper found at and released by synapses. It is possible that the prion protein could be released along with copper bound to it. Alternatively, copper could be captured by PrP on membranes on either side of the synpatic cleft. However, studies on the effects of copper on synapses suggest that the absence of prion protein expression makes synapse more susceptible to the deleterious effects of copper on neurotransmission implying that the prion protein is expressed there to have a protective role.

increase the amount of copper bound to PrPC isolated from the neurones. PrPC with two, three, or four copper atoms bound could protect neurones against the toxicity of superoxide, indicating that not only does the protein exhibit SOD activity in the test tube but it is also an effective antioxidant in culture.

Analysis of what happens when the PrPC binds other cations has shown that manganese can substitute for copper and that manganese-binding PrPC also has some SOD activity (67). However, this activity is rapidly lost and the manganese-binding protein undergoes a folding transition resulting in pro-tease-resistant protein. Such protein has similarities to the abnormal form of the protein PrP80. Although this resistant protein might not be infectious, this insight provides the intriguing possibility that the disease-specific form of the protein might be generated in vivo by incorporation of the wrong metal either as a result of dietary imbalance in metal ions or some other abnormality in metabolism of metals.

Changes in the amino acid sequence also influence the SOD activity of PrPC. Pure bread laboratory strains of mice usually express one of two alleles that differ only at two amino residues. A mouse strain that, as a general simplification, has a longer incubation time for the mouse form of scrapie prion disease expressed what is called the "b" allele of PrPC (69). Recombinant protein generated to have the sequence of the b allele has higher SOD activity than that of the normal allele (70). Additionally, this protein is more labile and breaks down more rapidly and loses activity more rapidly than the more common form of the mouse prion protein. These changes are reflected in the differences in the circular dichroism spectra of the proteins measured after aging the two proteins for several weeks (Fig. 4).

These findings suggest that PrPC is a copper-binding protein with antioxidant activity, expressed at the synapse (Fig. 5). A synaptic SOD may have benefits protecting synaptic termini from the damaging effects of superoxide and reactive oxygen species. Superoxide is known to inhibit some aspects of neurotransmission, and loss of synaptic spines is a common feature of diseases involving oxidative damage. Thus, as described earlier the reason for the high expression of the prion protein at the synapse may be the need to protect synaptic integrity.

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