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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 solubility due to ZnCl2 and CuCl2 is reversed by EDTA and SDS application.Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl2 (1 mM) and CuCl2 (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrPC signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.
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pone.0153931.g004: Reduced solubility due to ZnCl2 and CuCl2 is reversed by EDTA and SDS application.Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl2 (1 mM) and CuCl2 (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrPC signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.

Mentions: The effect of PrPC interaction with zinc, which results in a decrease in solubility, was reversible by treatment with ethylene diamine tetraacetic acid (EDTA) and sodium dodecylsulphate (SDS) (Fig 4). Mouse and bovine homogenates pre-incubated with zinc or copper ions were treated with the metal chelator EDTA. EDTA at equimolar and higher concentrations reversed PrPC from low to high solubility. Reversibility was determined using the amino terminal region-binding antibody SAF34 and the core region-binding antibody SAF70 as well (not shown). High solubility of zinc and copper-bound PrPC was also induced by treatment with the protein denaturing detergent sodium dodecylsulphate (SDS).


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 solubility due to ZnCl2 and CuCl2 is reversed by EDTA and SDS application.Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl2 (1 mM) and CuCl2 (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrPC signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153931.g004: Reduced solubility due to ZnCl2 and CuCl2 is reversed by EDTA and SDS application.Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl2 (1 mM) and CuCl2 (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrPC signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.
Mentions: The effect of PrPC interaction with zinc, which results in a decrease in solubility, was reversible by treatment with ethylene diamine tetraacetic acid (EDTA) and sodium dodecylsulphate (SDS) (Fig 4). Mouse and bovine homogenates pre-incubated with zinc or copper ions were treated with the metal chelator EDTA. EDTA at equimolar and higher concentrations reversed PrPC from low to high solubility. Reversibility was determined using the amino terminal region-binding antibody SAF34 and the core region-binding antibody SAF70 as well (not shown). High solubility of zinc and copper-bound PrPC was also induced by treatment with the protein denaturing detergent sodium dodecylsulphate (SDS).

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