Two amyloidogenic apolipoprotein A-I mutants: a fluorometric approach

RAMELLA, N.; PRIETO, E. D.; RIMOLDI. O. J.; TRICERRI, M. A.

Buzios, Brazil

Congreso; VII Iberoamerican Congress of Biophysics; 2009

Sociedad Iberoamericana de Biofisica

Amyloidosis is characterized by extra cellular deposits of anomalous fibrillar proteins. Human apolipoprotein A-I (apoA-I) does not normally misfold, but certain single natural mutations favor its pathologic aggregation, as Lys107-0 and Arg26Glicine (Iowa).  Here we studied the folding of both mutants, as compared with Wt apoA-I. Analysis of chemical denaturation shows that Iowa and Lys107-0 are more unstable at both acidic and physiological pH respect to the Wt. Under acidic conditions, mutant denaturation is less cooperative, suggesting intermediate folding states. Thioflavin-T (ThT) fluorescence intensity test showed high yield of amyloid-like aggregation at pH 4 and 5 of the three tested proteins, minimal at pH 6 and almost none under neutral conditions. These ThT-binding products sediment under mild centrifugation. Protein misfolding is concentration-dependent, and can occur under diluted solutions. Fluorescence intensity was always higher for Lys107-0, but no significant differences were detected between Wt and Iowa. Proteins show different types of aggregates as observed by electron microscopy. It is well-known that heparin favors the aggregation of different amyloidogenic proteins. At acidic pH heparin binding induced a significant increase in scattering respect to proteins at neutral pH, suggesting an increment in particles size, which is higher for Lys107-0 without difference between Wt and Iowa. Altogether these data suggest that apoA-I is sensitive to acidic conditions, but Lys107-0 is more sensitive to the pH environment. A strong conformational shift occurs between pH 5 and 6, thus pI plays an important role in determining protein aggregation; nevertheless, this is not the only factor, as the Iowa variant, having and extra positive charge, behaves similar to Wt under this experimental set up, but induces neuroamiloidosis in patients.