Different amyloid aggregation of smooth muscles titin in vitro

Abstract

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2-7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300-500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8-10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.

Congo Red and amyloids: history and relationship

“…The non-specificity of CR as an amyloid dye was also shown in our in vitro studies [31]. We found that smooth muscle titin (SMT) from chicken gizzards can form amorphous aggregates in two different solutions containing: (i) 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl) aggregates) (Figure 2A) and (ii) 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly) aggregates) (Figure 2B).…”

“…Interestingly, SMT(KCl) and SMT(Gly) aggregates, dialysed against a solution containing 0.6 M KCl, 30 mM KH 2 PO 4 , 1 mM DTT and 0.1 M NaN 3 at pH 7.0, also had amyloid X-ray patterns (Figure 2C,D). In both cases, ‘yellow to apple-green birefringence under polarised light’ was absent [31]. In our in vitro study, we did not observe a characteristic shift in the CR maximum optical absorbance from 490 to 540 nm in any sample, indicating the binding of the dye to titin amyloids [31].…”

“…In both cases, ‘yellow to apple-green birefringence under polarised light’ was absent [31]. In our in vitro study, we did not observe a characteristic shift in the CR maximum optical absorbance from 490 to 540 nm in any sample, indicating the binding of the dye to titin amyloids [31].…”

“…We found that smooth muscle titin (SMT) from chicken gizzards can form amorphous aggregates in two different solutions containing: (i) 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl) aggregates) (Figure 2A) and (ii) 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly) aggregates) (Figure 2B). The amyloid natures of SMT(KCl) and SMT(Gly) aggregates were confirmed by X-ray diffraction [31]. CR-stained SMT(Gly) aggregates had ‘yellow to apple-green birefringence under polarised light’, while no such birefringence for SMT(KCl) aggregates was observed (Figure 2A,B).…”

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion

“…Previously, we have shown that 500-kDa SMT can form amorphous aggregates of a quaternary cross-β structure intrinsic to all amyloid aggregates [ 36 , 37 ]. We have also investigated the cytotoxicity of those aggregates [ 36 ].…”