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Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes.

Brim S, Groschup MH, Kuczius T - PLoS ONE (2016)

Bottom Line: Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility.This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions.PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

View Article: PubMed Central - PubMed

Affiliation: Institute for Hygiene, University of Münster, Robert Koch-Strasse 41, 48149 Münster, Germany.

ABSTRACT
Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

No MeSH data available.


Related in: MedlinePlus

The PrPC–Zn2+ phenotype dominated in PrPC interactions after simultaneous and serial CuCl2 and ZnCl2 incubation.(A) Brain homogenate proteins (10%) derived from the C57BL wild-type and T182N transgenic mice were suspended in TBS with OGP (2%) followed by simultaneous incubation with ZnCl2 (Zn) and CuCl2 (Cu) in concentrations of 100 and 1000 µM as indicated. Proteins were separated by centrifugation into a protein fraction of high solubility represented as supernatant (S) and low solubility PrPC in the pellet (P). (B)Aliquots of highly soluble proteins from C57BL wild-type and T182N transgenic mice were pre-treated with zinc (Zn) and copper (Cu) ions (1 mM) each. As a result of centrifugation, proteins were separated into a pellet fraction (P1) and into highly soluble protein fractions, which were incubated additionally with ZnCl2 (+ Zn) and CuCl2 (+ Cu; 1 mM each) or in the absence of ions (-) as controls. Highly soluble proteins were detected in the supernatant S2, whereas poorly soluble PrPC were detected in the pellet P2 after centrifugation. Copper ions were unable to alter the PrPC–Zn2+ phenotype, which would have shown a conversion of pelleted PrPC to a fraction of highly soluble proteins. Protein suspensions were separated by SDS-PAGE, immunoblotted and PrPC were detected using mabs SAF34 and SAF70 as indicated. Signals were visualized after chemiluminescence reaction on an imager.
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pone.0153931.g006: The PrPC–Zn2+ phenotype dominated in PrPC interactions after simultaneous and serial CuCl2 and ZnCl2 incubation.(A) Brain homogenate proteins (10%) derived from the C57BL wild-type and T182N transgenic mice were suspended in TBS with OGP (2%) followed by simultaneous incubation with ZnCl2 (Zn) and CuCl2 (Cu) in concentrations of 100 and 1000 µM as indicated. Proteins were separated by centrifugation into a protein fraction of high solubility represented as supernatant (S) and low solubility PrPC in the pellet (P). (B)Aliquots of highly soluble proteins from C57BL wild-type and T182N transgenic mice were pre-treated with zinc (Zn) and copper (Cu) ions (1 mM) each. As a result of centrifugation, proteins were separated into a pellet fraction (P1) and into highly soluble protein fractions, which were incubated additionally with ZnCl2 (+ Zn) and CuCl2 (+ Cu; 1 mM each) or in the absence of ions (-) as controls. Highly soluble proteins were detected in the supernatant S2, whereas poorly soluble PrPC were detected in the pellet P2 after centrifugation. Copper ions were unable to alter the PrPC–Zn2+ phenotype, which would have shown a conversion of pelleted PrPC to a fraction of highly soluble proteins. Protein suspensions were separated by SDS-PAGE, immunoblotted and PrPC were detected using mabs SAF34 and SAF70 as indicated. Signals were visualized after chemiluminescence reaction on an imager.

Mentions: In the next series of experiments, we analysed the solubility of PrPC following combined and serial incubations with copper and zinc ions (Fig 6). The Zn2+-PrPC-phenotype predominated, even following simultaneous incubation with both copper and zinc ions in identical concentrations (Fig 6A). The three isoforms—un-, mono- and diglycosylated PrPC—were abundant in the pellet fraction when using mab SAF34 as a detection antibody. The full-length diglycosylated PrPC from wild-type mice and the full-length monoglycosylated derived from the mutant T182N mice showed high signal intensity in the pellet, whereas the truncated glycosylated C1 fragment and the unglycosylated wild-type PrPC isoforms dominated the supernatant when using mab SAF70. This Zn2+-PrPC-phenotype persisted as well in samples that had been treated serially with different metal ions (Fig 6B). Using the antibodies mab SAF34 and mab SAF70, an increase in the abundance of copper loaded PrPC was observed in the pellets after Zn2+ ions had been independently added to the proteins. Collectively, zinc binding to PrPC had an important influence on decreasing the solubility of PrPC, whereas copper binding failed to neutralize the Zn2+-PrPC-phenotype. This suggests an additional metal-protein interaction and a different biochemical characteristic independent of the copper-PrPC interactions.


Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes.

Brim S, Groschup MH, Kuczius T - PLoS ONE (2016)

The PrPC–Zn2+ phenotype dominated in PrPC interactions after simultaneous and serial CuCl2 and ZnCl2 incubation.(A) Brain homogenate proteins (10%) derived from the C57BL wild-type and T182N transgenic mice were suspended in TBS with OGP (2%) followed by simultaneous incubation with ZnCl2 (Zn) and CuCl2 (Cu) in concentrations of 100 and 1000 µM as indicated. Proteins were separated by centrifugation into a protein fraction of high solubility represented as supernatant (S) and low solubility PrPC in the pellet (P). (B)Aliquots of highly soluble proteins from C57BL wild-type and T182N transgenic mice were pre-treated with zinc (Zn) and copper (Cu) ions (1 mM) each. As a result of centrifugation, proteins were separated into a pellet fraction (P1) and into highly soluble protein fractions, which were incubated additionally with ZnCl2 (+ Zn) and CuCl2 (+ Cu; 1 mM each) or in the absence of ions (-) as controls. Highly soluble proteins were detected in the supernatant S2, whereas poorly soluble PrPC were detected in the pellet P2 after centrifugation. Copper ions were unable to alter the PrPC–Zn2+ phenotype, which would have shown a conversion of pelleted PrPC to a fraction of highly soluble proteins. Protein suspensions were separated by SDS-PAGE, immunoblotted and PrPC were detected using mabs SAF34 and SAF70 as indicated. Signals were visualized after chemiluminescence reaction on an imager.
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Related In: Results  -  Collection

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pone.0153931.g006: The PrPC–Zn2+ phenotype dominated in PrPC interactions after simultaneous and serial CuCl2 and ZnCl2 incubation.(A) Brain homogenate proteins (10%) derived from the C57BL wild-type and T182N transgenic mice were suspended in TBS with OGP (2%) followed by simultaneous incubation with ZnCl2 (Zn) and CuCl2 (Cu) in concentrations of 100 and 1000 µM as indicated. Proteins were separated by centrifugation into a protein fraction of high solubility represented as supernatant (S) and low solubility PrPC in the pellet (P). (B)Aliquots of highly soluble proteins from C57BL wild-type and T182N transgenic mice were pre-treated with zinc (Zn) and copper (Cu) ions (1 mM) each. As a result of centrifugation, proteins were separated into a pellet fraction (P1) and into highly soluble protein fractions, which were incubated additionally with ZnCl2 (+ Zn) and CuCl2 (+ Cu; 1 mM each) or in the absence of ions (-) as controls. Highly soluble proteins were detected in the supernatant S2, whereas poorly soluble PrPC were detected in the pellet P2 after centrifugation. Copper ions were unable to alter the PrPC–Zn2+ phenotype, which would have shown a conversion of pelleted PrPC to a fraction of highly soluble proteins. Protein suspensions were separated by SDS-PAGE, immunoblotted and PrPC were detected using mabs SAF34 and SAF70 as indicated. Signals were visualized after chemiluminescence reaction on an imager.
Mentions: In the next series of experiments, we analysed the solubility of PrPC following combined and serial incubations with copper and zinc ions (Fig 6). The Zn2+-PrPC-phenotype predominated, even following simultaneous incubation with both copper and zinc ions in identical concentrations (Fig 6A). The three isoforms—un-, mono- and diglycosylated PrPC—were abundant in the pellet fraction when using mab SAF34 as a detection antibody. The full-length diglycosylated PrPC from wild-type mice and the full-length monoglycosylated derived from the mutant T182N mice showed high signal intensity in the pellet, whereas the truncated glycosylated C1 fragment and the unglycosylated wild-type PrPC isoforms dominated the supernatant when using mab SAF70. This Zn2+-PrPC-phenotype persisted as well in samples that had been treated serially with different metal ions (Fig 6B). Using the antibodies mab SAF34 and mab SAF70, an increase in the abundance of copper loaded PrPC was observed in the pellets after Zn2+ ions had been independently added to the proteins. Collectively, zinc binding to PrPC had an important influence on decreasing the solubility of PrPC, whereas copper binding failed to neutralize the Zn2+-PrPC-phenotype. This suggests an additional metal-protein interaction and a different biochemical characteristic independent of the copper-PrPC interactions.

Bottom Line: Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility.This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions.PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

View Article: PubMed Central - PubMed

Affiliation: Institute for Hygiene, University of Münster, Robert Koch-Strasse 41, 48149 Münster, Germany.

ABSTRACT
Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

No MeSH data available.


Related in: MedlinePlus