Association Properties of βB1- and βA3-Crystallins: Ability to Form Heterotetramers

Crystallins, which exist at extremely high concentrations in the eye lens, are structural proteins critical for transmitting and focusing light. The human lens contains α-, β-, and γ-crystallins, of which β-crystallins, taken as a whole, represent the greatest part. Seven different subtypes of β-crystallins have been identified in the human lens, four of which are relatively acidic (βA1, βA2, βA3, and βA4), and three of which are relatively basic (βB1, βB2, and βB3). β- and γ-crystallins, which have highly similar core sequences, form the βγ-crystallin superfamily. Structurally, both β- and γ-crystallins contain two homologous domains connected by a short connecting sequence. Each domain contains two Greek-key motifs, and each motif comprises four antiparallel β-sheets (1). β-Crystallins contain N-terminal extensions ranging in length from 12 to 57 amino acids. In addition, the basic β-crystallins contain C-terminal extensions ranging from 11 to 16 amino acids. β-Crystallins are highly conserved across species with βA3-crystallin being over 95% identical between mouse and human. In contrast to β-crystallins, β-crystallins have either no terminal extensions or short extensions containing only a few amino acids (2). β-Crystallins are known to associate into dimers, tetramers, and higher-order complexes in vivo. Size-exclusion chromatography of bovine or human lens extracts shows three size classes of β-crystallin complexes: βH (160-200 kDa, primarily octamers), βL1 (70-100 kDa, primarily tetramers), and βL2 (46-50 kDa, primarily dimers) (3;4). The distribution of β-crystallins among different size classes is dependent on protein concentration, pH, and ionic strength (5;6). Despite the sequence homology between β- and γ-crystallins, γ-crystallins are observed in the lens strictly as monomers, which have prompted investigations into the role of terminal extensions in crystallin association. Previous studies have shown that although the terminal arms of β-crystallins are not required for association, the N-terminal extension of βA3-crystallin assists in self-association into homodimers, while the N-terminal extension of βB2-crystallin actually decreases association into homodimers (7;8). Several studies have also demonstrated a correlation between the length of the N-terminal extension of βB1-crystallin in lens extracts and the size class in which it migrates on size exclusion chromatography (9-11). Bateman et al. have previously shown that a truncated form of βB1- and βA3-crystallin migrate at a size of approximately 100 kDa on gel permeation chromatography, and have a similar size as estimated by laser light scattering.(12) While β-crystallins are known to form oligomers in vivo, a significant level of tetramers or higher-order complexes generally has not been observed in previous in vitro studies. This can be explained in part by the lower protein concentration being examined in vitro, compared with the extremely high concentration in the eye lens. It has previously been demonstrated that both mouse βB2-crystallin and human βA3-crystallin form reversible homodimers when present alone, and that these readily form heterodimers upon mixing and incubation at room temperature in the absence of denaturants (13). However, no heterotetramers or higher-order complexes were observed between these two crystallins. The current study employs a similar strategy to study the association properties of another pair of β-crystallins—mouse βB1- and human βA3-crystallins. When present alone, each protein associates into homodimers but neither forms homotetramers. Upon mixing in the absence of denaturants, heterocomplex formation between these two β-crystallin subtypes was observed by size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to the previous results obtained with βB2- and βA3-crystallins, both size-exclusion chromatography and sedimentation equilibrium of the mixture of βB1- and βA3-crystallins show a dimer-tetramer equilibrium, with a Kd of 1.1 μM calculated from the latter. These results show that mixed βB1- and βA3-crystallins associate predominantly into heterotetramers in vitro.