Interactions between small heat shock protein alpha-crystallin and galectin-related interfiber protein (GRIFIN) in the ocular lens.
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.
Barton
Kelly A
KA
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Hsu
Cheng-Da
CD
Petrash
J Mark
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011485:Protein Binding; D053719:Tandem Mass Spectrometry; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D038201:alpha-Crystallins