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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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Reactivity of oligomer-specific antibodies.A, the specificity of antibodies, purified as described in the legend to Fig. 7 from the antisera obtained after immunization with stabilized cystatin C oligomers, was tested by double radial immunodiffusion. L68Q cystatin C dimers, associated with hereditary cystatin C amyloid angiopathy (42), showed antigenic identity with stabilized dimers, whereas monomers where nonreactive. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison. B, immunoblots displaying the antigenic recognition of the oligomer-specific antibodies. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison.
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Figure 8: Reactivity of oligomer-specific antibodies.A, the specificity of antibodies, purified as described in the legend to Fig. 7 from the antisera obtained after immunization with stabilized cystatin C oligomers, was tested by double radial immunodiffusion. L68Q cystatin C dimers, associated with hereditary cystatin C amyloid angiopathy (42), showed antigenic identity with stabilized dimers, whereas monomers where nonreactive. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison. B, immunoblots displaying the antigenic recognition of the oligomer-specific antibodies. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison.

Mentions: The successful purification and the evident stability of the isolated oligomers prompted us to immunize rabbits in an effort to raise antisera specific for oligomeric cystatin C. We chose the stabilized and isolated dimers and HMW-oligomers, respectively, as immunogens for immunization trials in two sets of rabbits, as described under “Experimental Procedures.” The specific antigen-antibody reactions of the antisera obtained were characterized by diffusion in-gel techniques. To obtain oligomer-specific antibodies without reactivity toward monomeric cystatin C, we purified the antibodies from dimer and HMW-oligomer immunized animals according to the scheme in Fig. 7. First, absorption of monomer-binding antibodies was accomplished by affinity chromatography of immunoglobulin fractions using Sepharose-coupled monomeric cystatin C. The flow-through fractions were collected, and the oligomer reactivity was verified (Fig. 8) before isolation of oligomer-specific antibodies using stabilized HMW-oligomers coupled to a Sepharose column. The fractions obtained by acidic elution from this column contained oligomer-specific antibodies from either dimer or HMW-oligomer-immunized rabbits.


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)

Reactivity of oligomer-specific antibodies.A, the specificity of antibodies, purified as described in the legend to Fig. 7 from the antisera obtained after immunization with stabilized cystatin C oligomers, was tested by double radial immunodiffusion. L68Q cystatin C dimers, associated with hereditary cystatin C amyloid angiopathy (42), showed antigenic identity with stabilized dimers, whereas monomers where nonreactive. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison. B, immunoblots displaying the antigenic recognition of the oligomer-specific antibodies. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Reactivity of oligomer-specific antibodies.A, the specificity of antibodies, purified as described in the legend to Fig. 7 from the antisera obtained after immunization with stabilized cystatin C oligomers, was tested by double radial immunodiffusion. L68Q cystatin C dimers, associated with hereditary cystatin C amyloid angiopathy (42), showed antigenic identity with stabilized dimers, whereas monomers where nonreactive. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison. B, immunoblots displaying the antigenic recognition of the oligomer-specific antibodies. Polyclonal antibodies raised against monomeric cystatin C (anti-cystatin C pAb) were used for comparison.
Mentions: The successful purification and the evident stability of the isolated oligomers prompted us to immunize rabbits in an effort to raise antisera specific for oligomeric cystatin C. We chose the stabilized and isolated dimers and HMW-oligomers, respectively, as immunogens for immunization trials in two sets of rabbits, as described under “Experimental Procedures.” The specific antigen-antibody reactions of the antisera obtained were characterized by diffusion in-gel techniques. To obtain oligomer-specific antibodies without reactivity toward monomeric cystatin C, we purified the antibodies from dimer and HMW-oligomer immunized animals according to the scheme in Fig. 7. First, absorption of monomer-binding antibodies was accomplished by affinity chromatography of immunoglobulin fractions using Sepharose-coupled monomeric cystatin C. The flow-through fractions were collected, and the oligomer reactivity was verified (Fig. 8) before isolation of oligomer-specific antibodies using stabilized HMW-oligomers coupled to a Sepharose column. The fractions obtained by acidic elution from this column contained oligomer-specific antibodies from either dimer or HMW-oligomer-immunized rabbits.

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