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Stabilization, characterization, and selective removal of cystatin C amyloid oligomers.

Östner G, Lindström V, Hjort Christensen P, Kozak M, Abrahamson M, Grubb A - J. Biol. Chem. (2013)

Bottom Line: The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping.After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained.These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden.

ABSTRACT
The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

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Selectivity of oligomer-specific antibodies as shown by agarose gel electrophoresis. The oligomer-specific antibodies were added to isolated monomers, dimers, or a mixture of monomers and dimers to assess their antigenic specificity. Ab, oligomer-specific antibodies; M, stabilized monomer; D, stabilized dimer; MD, mixture of stabilized monomers and dimers. An asterisk marks the point of the sample application and the anode is marked by a plus sign.
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Figure 9: Selectivity of oligomer-specific antibodies as shown by agarose gel electrophoresis. The oligomer-specific antibodies were added to isolated monomers, dimers, or a mixture of monomers and dimers to assess their antigenic specificity. Ab, oligomer-specific antibodies; M, stabilized monomer; D, stabilized dimer; MD, mixture of stabilized monomers and dimers. An asterisk marks the point of the sample application and the anode is marked by a plus sign.

Mentions: To assess the reactivity of the oligomer-specific antibodies in free solutions of dimeric and monomeric cystatin C, the following system was used. Mixtures of antibodies, stabilized monomers, and stabilized dimers were prepared and electrophoresed in agarose gels under native conditions. The results showed that dimers were the preferred antigen, although a minor monomer reactivity could be detected. As shown in Fig. 9, the antibodies could deplete a solution of dimeric cystatin C. Addition of the antibodies to a solution of both monomeric and dimeric cystatin C resulted in a shift of the dimer/monomer ratio from 60/40 in the control sample to 15/85, indicating the potential of these antibodies for selective removal of dimers and, probably, oligomers in general.


Stabilization, characterization, and selective removal of cystatin C amyloid oligomers.

Östner G, Lindström V, Hjort Christensen P, Kozak M, Abrahamson M, Grubb A - J. Biol. Chem. (2013)

Selectivity of oligomer-specific antibodies as shown by agarose gel electrophoresis. The oligomer-specific antibodies were added to isolated monomers, dimers, or a mixture of monomers and dimers to assess their antigenic specificity. Ab, oligomer-specific antibodies; M, stabilized monomer; D, stabilized dimer; MD, mixture of stabilized monomers and dimers. An asterisk marks the point of the sample application and the anode is marked by a plus sign.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675580&req=5

Figure 9: Selectivity of oligomer-specific antibodies as shown by agarose gel electrophoresis. The oligomer-specific antibodies were added to isolated monomers, dimers, or a mixture of monomers and dimers to assess their antigenic specificity. Ab, oligomer-specific antibodies; M, stabilized monomer; D, stabilized dimer; MD, mixture of stabilized monomers and dimers. An asterisk marks the point of the sample application and the anode is marked by a plus sign.
Mentions: To assess the reactivity of the oligomer-specific antibodies in free solutions of dimeric and monomeric cystatin C, the following system was used. Mixtures of antibodies, stabilized monomers, and stabilized dimers were prepared and electrophoresed in agarose gels under native conditions. The results showed that dimers were the preferred antigen, although a minor monomer reactivity could be detected. As shown in Fig. 9, the antibodies could deplete a solution of dimeric cystatin C. Addition of the antibodies to a solution of both monomeric and dimeric cystatin C resulted in a shift of the dimer/monomer ratio from 60/40 in the control sample to 15/85, indicating the potential of these antibodies for selective removal of dimers and, probably, oligomers in general.

Bottom Line: The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping.After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained.These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden.

ABSTRACT
The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

Show MeSH
Related in: MedlinePlus