Biophysical Characterization of Lithostathine

Abstract

Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg 11 -Ile 12 peptide bond. It is deduced that the NH 2 -terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions.

High-speed atomic force microscopy coming of age

“…protease-K-resistant fibrils [181] that deposit in the brain of patients with Creutzfeld-Jakob disease [182] and Gertsmann-Straüssler-Scheinker syndrome [183], especially during the very early stages. figure 14) and (2) fusion of a growing protofibril to a pre-existing profofibril (light-blue arrows in figure 14).…”

A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation

“…In both species thread proteins were isolated and shown to be fibrillar by negative staining in the electron microscope (22,23). Purified PSP/lithostathine has been shown to form similar fibrils after tryptic cleavage in vitro (43), and prolonged incubation of PSP, purified by column chromatography, suggested that activation may occur by autoproteolysis. The studies reported here further extend the existence of fibrillar thread structures among pancreatic secretory proteins to include not only PTP but also PATP I and PATP III isoforms.…”

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

“…Recombinant Lithostathine-Lithostathine was prepared as described previously (7). In brief, recombinant lithostathine was produced in Chinese hamster ovary cells and purified on an immunoaffinity column.…”

“…We have studied its physical characteristics and observed that it readily polymerizes into fibrils after self-proteolysis of its N-terminal undecapeptide (7). On the basis of the x-ray structure of the monomer (8) and high resolution electron microscopy, we showed that fibrils formed a quadruple-helical filament (QHF-litho 1 ; see Ref.…”