Abstract
Purpose: :
Our previous results have shown that aggregates in aging human lenses contained crystallin fragments (Exp. Eye Res, 87:356-366, 2008; J. Biol. Chem., 279:10901-10909, 2004). The purpose of the present study was to determine whether in vivo-existing human lens crystallin fragments form aggregates in vitro with amyloidogenic properties.
Methods: :
Four fractions containing crystallin fragments [Fraction I (containing ~3.5 kDa species), Fraction II (containing ~3.5 to 7 kDa species), Fraction III (containing ~7 -10 kDa species) and Fraction IV ( containing >10 - 18 kDa species)] were isolated from water soluble fraction of human lenses of 50-70 year-old donors by a preparative SDS-PAGE method. The identity of crystallin fragments of Fractions I to IV was identified following 2D-gel electrophoretic separation and QTRAP mass spectrometric analysis. The fragments formed aggregates on in vitro incubation, which were isolated by a size-exclusion HPLC, and were analyzed for amyloid formation by assay with either Congo Red (CR) or Thioflavin (ThT). The aggregates were also analyzed for their molecular mass by a dynamic light scattering method and for their particle sizes and amyloid-type structures by transmission electron microscopic (TEM) method.
Results: :
Crystallin fragments from Fractions I to IV exhibited self-aggregated complexes with Mr of 5.5X105 to 1.0X108 Da. During CR assay, a red shift of the absorption band toward 540 nm was observed in the fragments of Fractions II and IV, whereas an increase in fluorescence was observed only in the Fraction III. During ThT assay, an increase in the fluorescence emission intensity at 490 nm was observed in Fractions II, III and IV suggested the amyloid-type complex formation. TEM images of Fractions II and III, showed amyloid-like structures (~100 nm) in the form of strings at low pH (2.0) and on heating at 60°C for 5 h.
Conclusions: :
The results suggested that in vivo-existing crystallin fragments formed amyloid-type of complexes during incubation in vitro.
Keywords: crystallins • proteomics • protein modifications-post translational