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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

Amino-terminally truncated PrPC isoforms are unable to bind metal ion.Proteins from pooled wild-type C57BL mouse brain homogenate were treated enzymatically with bromelain (50 µg/ml, 37°C, 60 min) generating a carboxy-terminal core protein lacking the octapeptide region to which divalent cations might bind. After incubation in the presence of CuCl2 (Cu), ZnCl2 (Zn) and MgCl2 (Mg) in concentrations of 1 mM each or in the absence of metal ions (no metal), proteins were separated by centrifugation into fractions of high and low solubility, respectively, represented as supernatants (S) and pellets (P). Proteins were separated by SDS-PAGE, immunoblotted and PrPC signals were visualized using mab SAF70 and chemiluminescence substrate development. Untreated protein (PrPC) was used as control.
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pone.0153931.g005: Amino-terminally truncated PrPC isoforms are unable to bind metal ion.Proteins from pooled wild-type C57BL mouse brain homogenate were treated enzymatically with bromelain (50 µg/ml, 37°C, 60 min) generating a carboxy-terminal core protein lacking the octapeptide region to which divalent cations might bind. After incubation in the presence of CuCl2 (Cu), ZnCl2 (Zn) and MgCl2 (Mg) in concentrations of 1 mM each or in the absence of metal ions (no metal), proteins were separated by centrifugation into fractions of high and low solubility, respectively, represented as supernatants (S) and pellets (P). Proteins were separated by SDS-PAGE, immunoblotted and PrPC signals were visualized using mab SAF70 and chemiluminescence substrate development. Untreated protein (PrPC) was used as control.

Mentions: Metal ions bind within the octarepeats at histidine residues with high affinity, whereas zinc binds with lower affinity [18;24]. To verify that the PrPC-Zn2+ phenotype is the result of metal binding to the octarepeats, we partially truncated the full-length protein by enzymatic treatment with bromelain obtained from Ananas comosus. The amino-terminal region was cleaved off, whereas a stable core protein consisting of glycosylated and nonglycosylated isoforms was generated (Fig 5). Removal of the amino-terminus prior to incubation in the presence of metals prevented PrPC from changing to low solubility. This result indicates an interaction of Zn2+ with the N-terminal region of PrPC.


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)

Amino-terminally truncated PrPC isoforms are unable to bind metal ion.Proteins from pooled wild-type C57BL mouse brain homogenate were treated enzymatically with bromelain (50 µg/ml, 37°C, 60 min) generating a carboxy-terminal core protein lacking the octapeptide region to which divalent cations might bind. After incubation in the presence of CuCl2 (Cu), ZnCl2 (Zn) and MgCl2 (Mg) in concentrations of 1 mM each or in the absence of metal ions (no metal), proteins were separated by centrifugation into fractions of high and low solubility, respectively, represented as supernatants (S) and pellets (P). Proteins were separated by SDS-PAGE, immunoblotted and PrPC signals were visualized using mab SAF70 and chemiluminescence substrate development. Untreated protein (PrPC) was used as control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153931.g005: Amino-terminally truncated PrPC isoforms are unable to bind metal ion.Proteins from pooled wild-type C57BL mouse brain homogenate were treated enzymatically with bromelain (50 µg/ml, 37°C, 60 min) generating a carboxy-terminal core protein lacking the octapeptide region to which divalent cations might bind. After incubation in the presence of CuCl2 (Cu), ZnCl2 (Zn) and MgCl2 (Mg) in concentrations of 1 mM each or in the absence of metal ions (no metal), proteins were separated by centrifugation into fractions of high and low solubility, respectively, represented as supernatants (S) and pellets (P). Proteins were separated by SDS-PAGE, immunoblotted and PrPC signals were visualized using mab SAF70 and chemiluminescence substrate development. Untreated protein (PrPC) was used as control.
Mentions: Metal ions bind within the octarepeats at histidine residues with high affinity, whereas zinc binds with lower affinity [18;24]. To verify that the PrPC-Zn2+ phenotype is the result of metal binding to the octarepeats, we partially truncated the full-length protein by enzymatic treatment with bromelain obtained from Ananas comosus. The amino-terminal region was cleaved off, whereas a stable core protein consisting of glycosylated and nonglycosylated isoforms was generated (Fig 5). Removal of the amino-terminus prior to incubation in the presence of metals prevented PrPC from changing to low solubility. This result indicates an interaction of Zn2+ with the N-terminal region of PrPC.

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