Binding of dexamethasone by alpha-crystallin.
OBJECTIVE
Long-term steroid therapy is a known risk factor for the development of posterior subcapsular cataract. Previous work in this laboratory has found soluble lens proteins to bind dexamethasone, but this binding is not due to a glucocorticoid receptor. This study was undertaken to identify the soluble protein or proteins involved in lens glucocorticoid binding.


METHODS
Bovine lens extract was incubated with 5.2 x 10(-)(8) M [(3)H]-dexamethasone for 3 hours, and the distribution of label assessed in the soluble and insoluble fractions after centrifugation. Soluble lens extract was fractionated using gel permeation chromatography to isolate and identify proteins involved in the binding. Total lens proteins, high-molecular-weight proteins, or alpha-crystallin were exposed to dexamethasone and the protein bound steroid measured after separation of free and bound ligand on a gel chromatography column. Scatchard analysis was used to determine dexamethasone-binding parameters. Sequence comparisons between bovine alphaA- and alphaB-crystallins and glucocorticoid-binding proteins were performed using a sequence-alignment program.


RESULTS
Of the total dexamethasone bound in lens extract, soluble proteins were found to account for 52%. The majority of the soluble protein-bound dexamethasone coeluted with the high-molecular-weight proteins that consisted mainly of alpha-crystallin. Binding studies with isolated proteins showed that alpha-crystallin accounted for more than 98% of total soluble dexamethasone binding in the lens. Scatchard analysis of steroid binding showed it to be a nonspecific partitioning event. Sequence comparisons between alphaA- and alphaB-crystallins and various glucocorticoid-binding proteins showed the lens proteins to have three regions of sequence homology with yeast corticosteroid-binding protein.


CONCLUSIONS
alpha-Crystallin is the principal soluble glucocorticoid binding protein in the lens. The steroid association is described by nonspecific partitioning and may be related to the unique structural characteristics of the protein. The nonspecific association with alpha-crystallin is not thought to be functional; however, it may aid in the increased covalent steroid modification observed for this protein.
Jobling
A I
AI
National Vision Research Institute of Australia, 386 Cardigan Street, Carlton, Victoria 3053, Australia.
Stevens
A
A
Augusteyn
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism*; D005938:Glucocorticoids / Q000378:metabolism*; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012995:Solubility
D003459:Crystallins; D005938:Glucocorticoids; D050886:HSP20 Heat-Shock Proteins; C494382:HSPB6 protein, human; D006360:Heat-Shock Proteins; D010750:Phosphoproteins; D003907:Dexamethasone