Interactions between Small Heat Shock Protein α-Crystallin and Galectin-Related Interfiber Protein (GRIFIN) in the Ocular Lens

TLDR: Results from cross-linking studies in transgenic mouse lenses designed for overexpression of His-tagged human alphaA-crystallin support the hypothesis that GRIFIN is a novel binding partner of alpha- Crystallin in the lens.

Abstract: The mammalian lens contains a very high concentration of soluble protein, estimated to be approximately 350 mg/mL, to provide refractive power. The majority of lens proteins belong to one of three major groups, which are designated α-, β-, and γ-crystallins (1). The α-crystallins, which constitute ~30% of lens protein, are heteromeric oligomers composed of two gene products, αA and αB, present in a 3:1 molar ratio in human lenses. The two ~20 kDa gene products assemble into oligomers with an average size of 600 kDa, indicating each complex contains approximately 30 monomers (2). The quaternary structure of α-crystallin is very dynamic due to the ability of αA- and αB-crystallin subunits to exchange between complexes in a temperature and time-dependent manner (3, 4). Like other members of the small heat shock protein (sHSP)1 family, α-crystallin has a chaperone-like activity defined by the ability to bind denatured or unstable proteins and prevent their aggregation. While sHSPs have minimal, if any, capacity to actively refold proteins, current evidence from in vitro studies suggests that they can transfer captured protein substrates to conventional chaperones for ATP-dependent refolding (5, 6). Mice with targeted disruption of the αA gene develop early cataracts caused by accumulation of light scattering protein aggregates (7), consistent with the view that a critical role for α-crystallin in the lens is maintenance of transparency. However, the mechanisms by which α-crystallin recognizes unstable and/or unfolded proteins are still unknown. Recent studies demonstrate that α-crystallin can interact with a wide variety of proteins involved with signaling and cytoskeletal structure (8). α-Crystallin interacts with various components of the cytoskeleton, including actin, vimentin, CP49, and filensin, but the effect this confers is variable (9–12). α-Crystallin stabilizes actin filaments both in vivo and in vitro when subjected to stresses, including heat and cytochalasin D (11, 12). However, the interaction with type III intermediate filament monomers prevents their assembly (10). α-Crystallin also associates with plasma membranes from lens fiber cells, and the total amount bound increases with age, diabetes, and cataract (13, 14). In vitro analysis of this binding suggests the interaction is hydrophobic and not dependent on a specific type of lipid (15). Beyond these insoluble cellular structures, the soluble protein interactions of α-crystallin are largely unknown. In vitro data suggest that intermolecular interactions occur between α-crystallin and a variety of signaling proteins and metabolic enzymes under native conditions (8, 16); however, the physiological importance is difficult to gauge since most of the identified proteins are expressed in the lens in small amounts, if at all (8). Judging from in vitro studies, substrate binding is significantly enhanced by ATP (16), although little is known about the effect this may have on its ability to bind and/or refold captured proteins (16–18). The goal of our study is to identify proteins that interact with α-crystallin in the context of the intact lens. As in previous studies which used epitope tagging to selectively enrich proteins that interact with HSP16.6 in the cyanobacterium Synechocystis sp. PCC 6803 (19), we have utilized a transgenic mouse model designed for lens-specific expression of His-tagged human αA-crystallin (20). Protein–protein interactions in the intact lens were fixed by a brief treatment with a cross-linking agent. α-Crystallin complexes that contain the bait transgene product were isolated by immobilized metal affinity chromatography (IMAC) for analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). As expected, the majority of interacting proteins captured by this approach were crystallins. Proteomic analysis of crystallin complexes isolated from transgenic lenses identified peptides derived from galectin-related interfiber protein (GRIFIN). Binding of GRIFIN by α-crystallin was confirmed using a filter-based binding assay. Dissociation constants and effects of ATP on binding are consistent with physiologically relevant interactions between α-crystallin and GRIFIN.