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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 glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl2 (Cu), ZnCl2 (Zn), MgCl2 (Mg) and CaCl2 (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrPC proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrPC proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrPC interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrPC changed into the pellet fraction.
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pone.0153931.g003: The glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl2 (Cu), ZnCl2 (Zn), MgCl2 (Mg) and CaCl2 (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrPC proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrPC proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrPC interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrPC changed into the pellet fraction.

Mentions: To analyze the influence of the glycosylation grade on the increase of pelleted PrPC-Zn2+ we added metal ions to wild-type mouse brain homogenates featuring two glycosylation sites, which can be modified at one or both sites and to mouse homogenates of the transgenic mouse line T182N characterized by one deleted glycosylation site [22] (Fig 3). In immunoblots the monoglycosylated band was observed as a strong signal, whereas the signal at the molecular mass of the unglycosylated PrPC was hardly visible. Additionally, an unknown fragment derived from the carboxy terminus was detected at approximately 22 kDa.


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 glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl2 (Cu), ZnCl2 (Zn), MgCl2 (Mg) and CaCl2 (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrPC proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrPC proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrPC interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrPC changed into the pellet fraction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153931.g003: The glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl2 (Cu), ZnCl2 (Zn), MgCl2 (Mg) and CaCl2 (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrPC proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrPC proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrPC interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrPC changed into the pellet fraction.
Mentions: To analyze the influence of the glycosylation grade on the increase of pelleted PrPC-Zn2+ we added metal ions to wild-type mouse brain homogenates featuring two glycosylation sites, which can be modified at one or both sites and to mouse homogenates of the transgenic mouse line T182N characterized by one deleted glycosylation site [22] (Fig 3). In immunoblots the monoglycosylated band was observed as a strong signal, whereas the signal at the molecular mass of the unglycosylated PrPC was hardly visible. Additionally, an unknown fragment derived from the carboxy terminus was detected at approximately 22 kDa.

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