title: Formation of amyloid fibrils in vitro by human gammaD-crystallin and its isolated domains.
abstract: OBJECTIVE
  Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Previous studies have shown that bovine crystallins form amyloid fibers under denaturing conditions and amyloid fibers accumulate in the lens of mice carrying mutations in crystallin genes. Within differentiating lens fiber cells, crystallins may be exposed to low pH lysosome compartments. We have investigated whether human gammaD-crystallin forms amyloid fibrils in vitro, when exposed to low pH partially denaturing conditions.
  
  
  METHODS
  Human gammaD-crystallin expressed and purified from E. coli, is stable and soluble at 37 degrees C, pH7, and refolds from the fully denatured state back to the native state under these conditions. Purified Human gammaD-crystallin as well as its isolated NH2- and COOH-terminal domains were incubated at acid pH and subsequently examined by transmission electron microscopy, absorption spectroscopy in the presence of Congo red, FTIR, and low-angle X-ray scattering.
  
  
  RESULTS
  Incubation of the intact protein at 37 degrees C in 50 mM acetate buffer pH 3 at 50 mg/ml for 2 days, led to formation of a viscous, gel-like solution. Examination of negatively stained samples by transmission electron microscopy revealed linear, non-branching fibrils of variable lengths, with widths ranging from 15 to 35 nm. Incubation with the dye Congo red generated the spectral red shift associated with dye binding to amyloid. Low-angle X-ray scattering from samples showed clear meridional reflection at 4.7 A and a more diffuse reflection on the equator between 10 and 11 A which is the typical "cross-beta" X-ray fiber diffraction pattern for amyloid fibers. FTIR was used to follow the evolution of the secondary structure of gammaD-crystallin with time during incubation of the protein at pH 3. The native protein displayed a major band at 1640 cm-1 that converted during incubation at 37 degrees C to a band at 1616 cm-1. An additional band at 1689 cm-1 also appeared with time. The presence of bands in the regions about 1620 cm-1 and about 1680 cm-1 has been attributed to the formation of intermolecular beta-sheet structure that characterizes the fibrillar amyloid motif. The isolated NH2-terminal 1-82 and COOH-terminal 86-174 domains of HgammaD-crystallin also formed amyloid fibrils after incubation under the same conditions, but to a lesser extent than the full length.
  
  
  CONCLUSIONS
  HgammaD-crystallin, as well as its isolated NH2-terminal 1-82 and COOH-terminal 86-174 domains of HgammaD-crystallin formed amyloid fibrils upon incubation at acid pH. Investigations of early stages in cataract formation within the lens will be required to assess whether amyloid fibrils play a role in the initiation of cataract in vivo.
authors.affiliation: Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece.
authors.forename: Katerina
authors.initials: K
authors.lastname: Papanikolopoulou
authors.forename: Ishara
authors.initials: I
authors.lastname: Mills-Henry
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Thol
authors.forename: Yongting
authors.initials: Y
authors.lastname: Wang
authors.forename: Abby A R
authors.initials: AA
authors.lastname: Gross
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Kirschner
authors.forename: Sean M
authors.initials: SM
authors.lastname: Decatur
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
mesh_terms: D000682:Amyloid / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D004396:Coloring Agents / Q000378:metabolism; D003224:Congo Red / Q000378:metabolism; D003459:Crystallins / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D017550:Spectroscopy, Fourier Transform Infrared; D014961:X-Ray Diffraction; D038222:gamma-Crystallins
chemical_list: D000682:Amyloid; C504903:CRYGD protein, human; D004396:Coloring Agents; D003459:Crystallins; D038222:gamma-Crystallins; D003224:Congo Red