ABSTRACT
Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent ß-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to ß-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an â¼2 cm(-1) shift in an apparent ß-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain ß-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible ß-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).
Subject(s)
Glycosylphosphatidylinositols/chemistry , Polysaccharides/chemistry , PrPSc Proteins/chemistry , Animals , Electrophoresis, Polyacrylamide Gel , Mice , Mice, Transgenic , Microscopy, Electron, Transmission , PrPSc Proteins/isolation & purification , Protein Conformation , Spectrophotometry, InfraredABSTRACT
Age-related cataract (ARC) is a multifactorial disease and the leading cause of blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. However, gene mutation studies on ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA (alphaA-crystallin) gene in three unrelated female sporadic cases among 711 ARC patients but not in 265 normal non-cataractous controls by SSCP and RFLP analysis. By comparing human recombinant wild-type and F71L-alphaA-crystallin, we characterized the functional significance of this missense mutation. Chromatography, fluorescence and far- and near-UV CD studies indicated that F71L missense mutation did not significantly affect the apparent molecular mass, secondary and tertiary structures and hydrophobicity of alphaA-crystallin. While the mutant alphaA-crystallin displayed significant (35-90%) loss of chaperone-like activity (CLA) in thermal aggregation of carbonic anhydrase, betaL- and gamma-crystallins, it showed moderate (10-50%) loss in CLA in DTT-induced aggregation of insulin and lysozyme. This is the first report of an alphaA-F71L mutation being associated with ARC and suggests that ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.
Subject(s)
Aging/genetics , Amino Acid Substitution/genetics , Cataract/genetics , Genetic Predisposition to Disease , Mutation/genetics , alpha-Crystallin A Chain/genetics , Base Sequence , Case-Control Studies , Chromatography, Gel , Circular Dichroism , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Exons/genetics , Female , Humans , Light , Male , Middle Aged , Molecular Sequence Data , Mutant Proteins , Protein Structure, Quaternary , Scattering, Radiation , Spectrometry, Fluorescence , Time Factors , Tryptophan , alpha-Crystallin A Chain/chemistryABSTRACT
As a member of the small heat shock protein superfamily, alpha-crystallin has a chaperone-like ability to recognize and bind denatured or unfolded proteins and prevent their aggregation. Recent studies suggest that alpha-crystallin may also interact with a variety of proteins under native conditions in vitro. To identify potential binding partners for alpha-crystallin in the intact ocular lens, we conducted cross-linking studies in transgenic mouse lenses designed for overexpression of His-tagged human alphaA-crystallin. Interacting proteins were copurified with the epitope-tagged crystallin complexes and were identified by tandem mass spectrometry. This approach identified GRIFIN (galectin-related interfiber protein) as a novel binding partner. Consistent with results from cross-linking, GRIFIN subunits copurified with alpha-crystallin complexes during size exclusion chromatography of nontransgenic mouse lens extracts prepared without chemical cross-linking. Equilibrium binding to GRIFIN was studied using native alpha-crystallin isolated from calf lenses as well as oligomeric complexes reconstituted from recombinant alphaA- and alphaB-crystallin subunits. Calf lens alpha-crystallin binds GRIFIN with relatively high affinity (K(d) = 6.5 +/- 0.8 microM) at a stoichiometry of 0.25 +/- 0.01 GRIFIN monomer/alpha-crystallin subunit. The binding interaction between alpha-crystallin and GRIFIN is enhanced up to 5-fold in the presence of 3 mM ATP. These binding data support the hypothesis that GRIFIN is a novel binding partner of alpha-crystallin in the lens.
