Limits...
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.

Show MeSH

Related in: MedlinePlus

Selective depletion of dimeric cystatin C in blood plasma. Quantification of cystatin C in the fractions of gel filtration of plasma samples on a column of Superdex 75 GL 10/300 (A and B). In the control sample (black line) stabilized dimeric cystatin C was added to normal human plasma, resulting in a concentration of 1.7 mg/liter (endogenous monomeric cystatin C = 0.9 mg/liter). The oligomer-specific antibodies were added to the plasma sample 2 h before fractionation (red and blue lines). B, plot displaying the percentage of dimeric cystatin C of the total cystatin C (monomeric + dimeric) after the addition of increasing amounts of oligomer-specific antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675580&req=5

Figure 10: Selective depletion of dimeric cystatin C in blood plasma. Quantification of cystatin C in the fractions of gel filtration of plasma samples on a column of Superdex 75 GL 10/300 (A and B). In the control sample (black line) stabilized dimeric cystatin C was added to normal human plasma, resulting in a concentration of 1.7 mg/liter (endogenous monomeric cystatin C = 0.9 mg/liter). The oligomer-specific antibodies were added to the plasma sample 2 h before fractionation (red and blue lines). B, plot displaying the percentage of dimeric cystatin C of the total cystatin C (monomeric + dimeric) after the addition of increasing amounts of oligomer-specific antibodies.

Mentions: To test if the oligomer-specific antibodies would selectively target oligomers also in complex biological systems, we developed a sensitive system for the detection of monomers and dimers in blood plasma using gel filtration followed by immunodetection (SEC-ELISA), as described under “Experimental Procedures.” The oligomer-specific antibodies were, in increasing concentrations, added to equal aliquots of plasma containing 0.9 mg/liter of monomeric and 1.7 mg/liter of dimeric cystatin C and, after a 2-h incubation at room temperature and centrifugation at 10,000 × g for 10 min, the mixtures were run in the SEC-ELISA system. A significant and dose-dependent decrease in cystatin C immunoreactivity in the fractions corresponding to dimers was observed, whereas monomeric cystatin C was less affected (Fig. 10). Using antibody concentrations of 8 and 16 mg/liter, respectively, corresponding to 50 and 100 nm, the dimeric cystatin C fraction was reduced by ∼65 and 95%, respectively (Fig. 10A). A concomitant increase in cystatin C immunoreactivity was also observed in the void volume of the column (SEC exclusion >100 kDa).


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)

Selective depletion of dimeric cystatin C in blood plasma. Quantification of cystatin C in the fractions of gel filtration of plasma samples on a column of Superdex 75 GL 10/300 (A and B). In the control sample (black line) stabilized dimeric cystatin C was added to normal human plasma, resulting in a concentration of 1.7 mg/liter (endogenous monomeric cystatin C = 0.9 mg/liter). The oligomer-specific antibodies were added to the plasma sample 2 h before fractionation (red and blue lines). B, plot displaying the percentage of dimeric cystatin C of the total cystatin C (monomeric + dimeric) after the addition of increasing amounts of oligomer-specific antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Selective depletion of dimeric cystatin C in blood plasma. Quantification of cystatin C in the fractions of gel filtration of plasma samples on a column of Superdex 75 GL 10/300 (A and B). In the control sample (black line) stabilized dimeric cystatin C was added to normal human plasma, resulting in a concentration of 1.7 mg/liter (endogenous monomeric cystatin C = 0.9 mg/liter). The oligomer-specific antibodies were added to the plasma sample 2 h before fractionation (red and blue lines). B, plot displaying the percentage of dimeric cystatin C of the total cystatin C (monomeric + dimeric) after the addition of increasing amounts of oligomer-specific antibodies.
Mentions: To test if the oligomer-specific antibodies would selectively target oligomers also in complex biological systems, we developed a sensitive system for the detection of monomers and dimers in blood plasma using gel filtration followed by immunodetection (SEC-ELISA), as described under “Experimental Procedures.” The oligomer-specific antibodies were, in increasing concentrations, added to equal aliquots of plasma containing 0.9 mg/liter of monomeric and 1.7 mg/liter of dimeric cystatin C and, after a 2-h incubation at room temperature and centrifugation at 10,000 × g for 10 min, the mixtures were run in the SEC-ELISA system. A significant and dose-dependent decrease in cystatin C immunoreactivity in the fractions corresponding to dimers was observed, whereas monomeric cystatin C was less affected (Fig. 10). Using antibody concentrations of 8 and 16 mg/liter, respectively, corresponding to 50 and 100 nm, the dimeric cystatin C fraction was reduced by ∼65 and 95%, respectively (Fig. 10A). A concomitant increase in cystatin C immunoreactivity was also observed in the void volume of the column (SEC exclusion >100 kDa).

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