muramidase and Cataract

We present a novel hypothesis for the molecular mechanism of autosomal dominant cataract linked to two mutations in the alphaA-crystallin gene of the ocular lens. AlphaA-crystallin is a molecular chaperone that plays a critical role in the suppression of protein aggregation and hence in the long term maintenance of lens optical properties. Using a steady state binding assay in which the chaperone-substrate complex is directly detected, we demonstrate that the mutations result in a substantial increase in the level of binding to non-native states of the model substrate T4 lysozyme. The structural basis of the enhanced binding is investigated through equivalent substitutions in the homologous heat shock protein 27. The mutations shift the oligomeric equilibrium toward a dissociated multimeric form previously shown to be the binding-competent state. In the context of a recent thermodynamic model of chaperone function that proposes the coupling of small heat shock protein activation to the substrate folding equilibrium (Shashidharamurthy, R., Koteiche, H. A., Dong, J., and McHaourab, H. S. (2005) J. Biol. Chem. 280, 5281-5289), the enhanced binding by the alphaA-crystallin mutants is predicted to shift the substrate folding equilibrium toward non-native intermediates, i.e. the mutants promote substrate unfolding. Given the high concentration of alphaA-crystallin in the lens, the molecular basis of pathogenesis implied by our results is a gain of function that leads to the binding of undamaged proteins and subsequent precipitation of the saturated alpha-crystallin complexes in the developing lens of affected individuals.

Topics: alpha-Crystallin A Chain; Anisotropy; Bacteriophage T4; Binding Sites; Cataract; Humans; Kinetics; Lens, Crystalline; Molecular Chaperones; Muramidase; Mutagenesis, Site-Directed; Mutation; Protein Binding; Substrate Specificity; Thermodynamics

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.

Topics: Animals; Cataract; Cattle; Crystallins; Glucosephosphate Dehydrogenase; Humans; In Vitro Techniques; Lens, Crystalline; Molecular Chaperones; Muramidase; Recombinant Proteins; Ultraviolet Rays

A remarkable resemblance between the appearance of opacity in lysozyme--salt water mixtures and the development of opacity in cold cataract in the young rat lens is strong evidence that cold cataract is fundamentally a phase separation of the "protein-water binary mixture" in the lens.

Topics: Animals; Cataract; Crystallins; Lens, Crystalline; Models, Biological; Muramidase; Rats; Sodium Chloride; Solutions; Thermodynamics

The photo-oxidative effect of sunlight on the tryptophan residues of proteins and on free tryptophan is described. Evidence is presented that the indole ring is split to yield N'-formylkynurenine. The possible relation of this photo-oxidative change to changes in the lens proteins of brown cataracts is discussed.

Topics: Animals; Cataract; Cattle; Chromatography; Crystallins; Electrophoresis; Fluorescence; Humans; In Vitro Techniques; Kynurenine; Muramidase; Oxidation-Reduction; Serum Albumin; Spectrophotometry; Sunlight; Tryptophan