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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

Interaction of metals with PrPC induced a change to low solubility.Immunoblot analysis of brain homogenates (10%) derived from C57BL wild-type mice, bovine, human and sheep is as indicated. Homogenates were pre-incubated with various metal ions (1 mM) followed by centrifugation to generate a protein fraction of high solubility in the supernatant and of low solubility in the pellet. The pellet fractions were re-suspended in homogenate buffer back to the original volume. Following the addition of sample buffer, proteins were denatured by heating, and identical volumes were loaded for separation on SDS-PAGE and subsequent immunoblotting. PrPC specific signals were detected on immunoblots by mab SAF34 and visualized using a chemiluminescence substrate. Unlike other metals, the interaction of ZnCl2 with PrPC led to reduced protein solubility in samples from all four species.
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pone.0153931.g001: Interaction of metals with PrPC induced a change to low solubility.Immunoblot analysis of brain homogenates (10%) derived from C57BL wild-type mice, bovine, human and sheep is as indicated. Homogenates were pre-incubated with various metal ions (1 mM) followed by centrifugation to generate a protein fraction of high solubility in the supernatant and of low solubility in the pellet. The pellet fractions were re-suspended in homogenate buffer back to the original volume. Following the addition of sample buffer, proteins were denatured by heating, and identical volumes were loaded for separation on SDS-PAGE and subsequent immunoblotting. PrPC specific signals were detected on immunoblots by mab SAF34 and visualized using a chemiluminescence substrate. Unlike other metals, the interaction of ZnCl2 with PrPC led to reduced protein solubility in samples from all four species.

Mentions: Homogenized brain tissue suspensions from human, bovine, sheep and mouse were treated with the detergent N-octyl-β-D-glucopyranoside (OGP), which is non-ionic, and does not denature proteins. After centrifugation, the highly soluble proteins in the supernatants were exposed to various metal ions prior to incubation. Although we exclusively used proteins with high solubility, the binding of zinc to PrPC induced a considerable shift of proteins into a PrPC isoform of low solubility retrievable in the pellet after high-speed centrifugation (Fig 1). The incubation of PrPC with copper ions clearly had a much lower effect on decreasing the solubility of PrPC under these conditions; the levels of PrPC with low solubility were only marginally higher than those observed in the presence of Co2+ and Ni2+. PrPC remained highly soluble in the presence of Ca2+, Mg2+ and Mn2+ ions as well as in the absence of metals.


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)

Interaction of metals with PrPC induced a change to low solubility.Immunoblot analysis of brain homogenates (10%) derived from C57BL wild-type mice, bovine, human and sheep is as indicated. Homogenates were pre-incubated with various metal ions (1 mM) followed by centrifugation to generate a protein fraction of high solubility in the supernatant and of low solubility in the pellet. The pellet fractions were re-suspended in homogenate buffer back to the original volume. Following the addition of sample buffer, proteins were denatured by heating, and identical volumes were loaded for separation on SDS-PAGE and subsequent immunoblotting. PrPC specific signals were detected on immunoblots by mab SAF34 and visualized using a chemiluminescence substrate. Unlike other metals, the interaction of ZnCl2 with PrPC led to reduced protein solubility in samples from all four species.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153931.g001: Interaction of metals with PrPC induced a change to low solubility.Immunoblot analysis of brain homogenates (10%) derived from C57BL wild-type mice, bovine, human and sheep is as indicated. Homogenates were pre-incubated with various metal ions (1 mM) followed by centrifugation to generate a protein fraction of high solubility in the supernatant and of low solubility in the pellet. The pellet fractions were re-suspended in homogenate buffer back to the original volume. Following the addition of sample buffer, proteins were denatured by heating, and identical volumes were loaded for separation on SDS-PAGE and subsequent immunoblotting. PrPC specific signals were detected on immunoblots by mab SAF34 and visualized using a chemiluminescence substrate. Unlike other metals, the interaction of ZnCl2 with PrPC led to reduced protein solubility in samples from all four species.
Mentions: Homogenized brain tissue suspensions from human, bovine, sheep and mouse were treated with the detergent N-octyl-β-D-glucopyranoside (OGP), which is non-ionic, and does not denature proteins. After centrifugation, the highly soluble proteins in the supernatants were exposed to various metal ions prior to incubation. Although we exclusively used proteins with high solubility, the binding of zinc to PrPC induced a considerable shift of proteins into a PrPC isoform of low solubility retrievable in the pellet after high-speed centrifugation (Fig 1). The incubation of PrPC with copper ions clearly had a much lower effect on decreasing the solubility of PrPC under these conditions; the levels of PrPC with low solubility were only marginally higher than those observed in the presence of Co2+ and Ni2+. PrPC remained highly soluble in the presence of Ca2+, Mg2+ and Mn2+ ions as well as in the absence of metals.

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