Probing the specific interactions of AlphaA- crystallin and its aging- and cataract-associated forms with lens cell membrane mimics

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ABSTRACT The human lens contains high concentrations of the molecular chaperones, alpha-crystallins (ACs), that maintain lens transparency. AC consists of alphaA- (AAC) and alphaB- (ABC) crystallins, both distinct proteins, broadly similar in structure and function. Upon aging and in age-related cataract (ARC), AC is bound to the lens fiber cell membrane which leads to the loss of transparency. It is unclear which of the two proteins (AAC or ABC) binds to the membrane, or if both do, and how; but the binding does promote opacity. Therefore, to eventually delay or mitigate lens opacity it is important to first determine the molecular basis underlying such binding. AAC is primarily lens specific − unlike ABC − and is present at about three times higher concentration. There is more evidence implicating AAC than ABC in direct membrane binding – therefore AAC is our focus here. The lens membrane is unique in composition, and contains elevated levels of sphingomyelin (SM), dihydrosphingomyelin (DSM), and cholesterol. Aging- related protein modifications and membrane compositional changes appear to result in a significant increase in ACs binding to the membrane, especially in the population of AAC-containing intramolecular disulfides (S-S). This raises the question: Do the well-known aging-related AAC modifications, i.e., (i) truncation of C-terminal Ser (AAC-1-172), and (ii) deamidation of Asn 101 (AAC-N101D), or (iii) its “pre- senile” cataract-associated G98R mutant, show increased membrane-binding relative to AAC? To distinguish these processes, we hypothesize that AAC binds to SM (and DSM), aided by other phospholipids (PLs), and modulated by cholesterol in the human lens membrane. The disulfide-oxidized form of AAC may play a role in this process. For clearly defining the role of individual membrane-lipid components, we will use liposomes (membrane mimics with defined compositions) instead of human lens membranes, and recombinant human crystallins. For a comprehensive understanding of the binding process, we will use targeted biophysical techniques: FTIR (for SM, cholesterol, and protein); Raman (for −SH and SS bonds and bond strength); CD (for protein conformation); solution NMR (for residue-specific structural information), and Langmuir balance (to measure lipid-monolayer insertion by AAC). We propose the following specific aims: 1. Characterize AAC binding to the membrane and define the role of, (i) lipid composition, and (ii) oxidized AAC in membrane binding. 2. Determine if (i) membrane binding increases in age-modified AAC forms (AAC-1-172, & AAC-N101D), and (ii) the “pre-senile” G98R mutant; and if (iii) the oxidized age-modified AAC forms play a role in membrane binding.