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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

Reduced PrPC solubility is an effect specific to the binding of certain metal ions.Protein suspensions from brain homogenates derived from C57BL wild-type mice were reacted with increasing concentrations of ZnCl2, CuCl2 and MgCl2 as indicated. Proteins were centrifuged resulting in a separation of PrPC into a fraction of high solubility in the supernatant and a fraction of low solubility in the pellet. Following SDS-PAGE and immunoblotting, PrPC signals were visualized using mabs SAF34 (A) and SAF70 (B) followed by chemiluminescence substrate development. PrPC specific bands are indicated with d (diglycosylated), m (monoglycosylated), n (nonglycosylated) and C1 (truncated glycosylated C1 fragment). Neuron-specific enolase (NSE) was used as control (C).
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pone.0153931.g002: Reduced PrPC solubility is an effect specific to the binding of certain metal ions.Protein suspensions from brain homogenates derived from C57BL wild-type mice were reacted with increasing concentrations of ZnCl2, CuCl2 and MgCl2 as indicated. Proteins were centrifuged resulting in a separation of PrPC into a fraction of high solubility in the supernatant and a fraction of low solubility in the pellet. Following SDS-PAGE and immunoblotting, PrPC signals were visualized using mabs SAF34 (A) and SAF70 (B) followed by chemiluminescence substrate development. PrPC specific bands are indicated with d (diglycosylated), m (monoglycosylated), n (nonglycosylated) and C1 (truncated glycosylated C1 fragment). Neuron-specific enolase (NSE) was used as control (C).

Mentions: To verify a zinc specific effect, mouse PrPC was incubated with increasing concentrations of the zinc ion (Fig 2). We found that ZnCl2 reduced PrPC solubility at concentrations as low as 50 to 100 µM, and insolubility rose as Zn2+ concentrations were increased. With respect to the protein profile in the pellet fraction, the full length diglycosylated isoforms dominated, whereas the truncated glycosylated C1 proteins and the unglycosylated isoforms exhibited lower intensities. Zn2+ bound PrPC was determined by using the monoclonal antibody SAF34 that recognizes an epitope within the octarepeats (Fig 2A). To analyze whether this banding profile originated from an antibody specific effect, we examined protein profiles by binding mab SAF70 to the core protein region (Fig 2B). Interestingly, two different banding patterns with Zn2+ interaction were revealed. In the supernatants, a dominant band was observed, which consisted of highly soluble PrPC having a molecular mass of unglycosylated PrPC. This protein signal resulted from an overlay of unglycosylated PrPC and truncated glycosylated C1 protein, because both isoforms had the same electrophoretic mobility. Unglycosylated PrPC demonstrated lower signal intensity than truncated glycosylated C1 protein shown by protein deglycosylation using N-glycosidase F treatment (data not shown). In contrast, low PrPC signals were observed for the full-length glycosylated isoforms. In the pellets, however, high signals were detected for the full-length diglycosylated PrPC band; these intensified as Zn2+ concentrations were increased. This result indicated that binding of Zn2+ to PrPC is directly correlated to the solubility reduction of the full-length diglycosylated form of PrPC. This differential solubility effect as a result of Zn2+-PrPC interaction was not observed with Cu2+ and Mg2+ under the same conditions and specificity was demonstrated by using the neuron specific enolase (NSE) as a control protein, which remained highly soluble within the range of Zn2+concentration used (Fig 2C).


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)

Reduced PrPC solubility is an effect specific to the binding of certain metal ions.Protein suspensions from brain homogenates derived from C57BL wild-type mice were reacted with increasing concentrations of ZnCl2, CuCl2 and MgCl2 as indicated. Proteins were centrifuged resulting in a separation of PrPC into a fraction of high solubility in the supernatant and a fraction of low solubility in the pellet. Following SDS-PAGE and immunoblotting, PrPC signals were visualized using mabs SAF34 (A) and SAF70 (B) followed by chemiluminescence substrate development. PrPC specific bands are indicated with d (diglycosylated), m (monoglycosylated), n (nonglycosylated) and C1 (truncated glycosylated C1 fragment). Neuron-specific enolase (NSE) was used as control (C).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836684&req=5

pone.0153931.g002: Reduced PrPC solubility is an effect specific to the binding of certain metal ions.Protein suspensions from brain homogenates derived from C57BL wild-type mice were reacted with increasing concentrations of ZnCl2, CuCl2 and MgCl2 as indicated. Proteins were centrifuged resulting in a separation of PrPC into a fraction of high solubility in the supernatant and a fraction of low solubility in the pellet. Following SDS-PAGE and immunoblotting, PrPC signals were visualized using mabs SAF34 (A) and SAF70 (B) followed by chemiluminescence substrate development. PrPC specific bands are indicated with d (diglycosylated), m (monoglycosylated), n (nonglycosylated) and C1 (truncated glycosylated C1 fragment). Neuron-specific enolase (NSE) was used as control (C).
Mentions: To verify a zinc specific effect, mouse PrPC was incubated with increasing concentrations of the zinc ion (Fig 2). We found that ZnCl2 reduced PrPC solubility at concentrations as low as 50 to 100 µM, and insolubility rose as Zn2+ concentrations were increased. With respect to the protein profile in the pellet fraction, the full length diglycosylated isoforms dominated, whereas the truncated glycosylated C1 proteins and the unglycosylated isoforms exhibited lower intensities. Zn2+ bound PrPC was determined by using the monoclonal antibody SAF34 that recognizes an epitope within the octarepeats (Fig 2A). To analyze whether this banding profile originated from an antibody specific effect, we examined protein profiles by binding mab SAF70 to the core protein region (Fig 2B). Interestingly, two different banding patterns with Zn2+ interaction were revealed. In the supernatants, a dominant band was observed, which consisted of highly soluble PrPC having a molecular mass of unglycosylated PrPC. This protein signal resulted from an overlay of unglycosylated PrPC and truncated glycosylated C1 protein, because both isoforms had the same electrophoretic mobility. Unglycosylated PrPC demonstrated lower signal intensity than truncated glycosylated C1 protein shown by protein deglycosylation using N-glycosidase F treatment (data not shown). In contrast, low PrPC signals were observed for the full-length glycosylated isoforms. In the pellets, however, high signals were detected for the full-length diglycosylated PrPC band; these intensified as Zn2+ concentrations were increased. This result indicated that binding of Zn2+ to PrPC is directly correlated to the solubility reduction of the full-length diglycosylated form of PrPC. This differential solubility effect as a result of Zn2+-PrPC interaction was not observed with Cu2+ and Mg2+ under the same conditions and specificity was demonstrated by using the neuron specific enolase (NSE) as a control protein, which remained highly soluble within the range of Zn2+concentration used (Fig 2C).

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