Under Conditions of Amyloid Formation Bovine Carbonic Anhydrase B Undergoes Fragmentation by Acid Hydrolysis

Abstract

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins’ aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II

“…Recently, more detailed description of morphological types of these fibrils has been published [ 25 ]. It was shown that a distinctive feature of the I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril ribbons, while the other mutant variants form worm-like, twisted, or single-long fibrils.…”

“…These results indicated that the observed amyloids are formed not of full-size proteins but of fragments of polypeptide chain derived via hydrolysis of the proteins. The detailed study of this mechanism of amyloid formation of carbonic anhydrase II is presented in recent paper [ 25 ]. Briefly, it was revealed that acid hydrolysis during incubation at pH 2.7 and 57 °C leads to the wild-type carbonic anhydrase cleavage into fragments of about 10 kDa and production of free and associated peptides.…”

“…The latter are homogeneous in molecular weight (about 200 kDa), preserve a pronounced secondary structure of the full-length protein, and act as precursors of amyloid fibrils. Additionally, it was shown that acid hydrolysis of the wild-type carbonic anhydrase is almost complete after 6-day incubation [ 25 ].…”

“…It allowed for demonstrating that the substitutions L78A and L139A caused the increase in lag time of amyloid growth (Figure5C), whereas M239A and I208A significantly affect the apparent rate constant of the process (Figure5D).Histograms 5C and 5D show that the protein with the I208A substitution differs from other mutated proteins in such parameters as lag time and rate constant.Electron microscopy studies confirmed that all the investigated proteins formed fibrillar structures after reaching the plateau (Figure6). However, differences in fibril shape for various mutant proteins can be seen in Figure6.Recently, more detailed description of morphological types of these fibrils has been published[25]. It was shown that a distinctive feature of the I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril ribbons, while the other mutant variants form worm-like, twisted, or single-long fibrils.Thus, both thioflavin T binding and electron microscopy data give evidence on formation of fibrillar structures during incubation of wild-type carbonic anhydrase and its mutant forms at pH 2.7 and 57 • C. Taking into account the studies of other groups[18,19], one can suppose that amyloid fibrils were also obtained in our work.Analysis of SDS-electrophoresis of carbonic anhydrase II incubated in acidic medium (pH 2.7) at elevated temperature (57 • C) showed that acid hydrolysis of this protein takes place under such conditions.…”