Interaction Between Motifs of Human βA3-Crystallin and A or B Crystallins

Abstract

Purpose: : The purpose of the study was to determine the regions of human βA3-crystallin that interact with αA or αB crystallins.

Methods: : The following three approaches were used to determine the regions of βA3-crystallin that interact with human αA or αB crystallins: (1) Mammalian two-hybrid method was used to examine interacting regions among crystallins. Briefly, five deletion mutants of βA3 were generated starting with deletion of N-terminal extension ([NTE], βA3-NTE), motif I (βA3-NTE+I), motif II (βA3-NTE+I+II), and motif III (βA3-III), and motif IV (βA3-III + IV) using specific primers and pMβA3- and pVP16βA3- plasmid DNA as templates. Wild-type (WT) αA and WT αB were subcloned into pM and pVP16 vectors. These constructs were co-transfected along with a SEAP reporter vector into HeLa cells using Lipofectamine 2000, and the SEAP activity was assayed using Great EscApe SEAP fluorescence detection kit. Cells were also examined for in situ co-localization of fusion proteins using either monoclonal or polyclonal antibodies and red or green fluorescent conjugated secondary antibodies. (2) The formations of oligomers between αA or αB and βA3 were analyzed by size-exclusion HPLC and the molecular masses were determined by Dynamic Light Scattering (DLS). (3) Sulfo SAED, a cross-linker was used to identify the cross-linked residues between αA:αB, αA:βA3, αB:βA3 following SDS-PAGE and mass spectrometric analyses.

Results: : The fold-increase of SEAP activity between WT βA3 and WT αA or WT αB compared to basal level suggested significant interaction among crystallins. The interactions between WT αA or WT αB and deletion mutants of βA3 showed a gradual decline with sequential deletion of βA3 regions suggesting involvement of each of the βA3 motifs. However, deletion of motif III plus IV resulted in maximum loss of interaction. HPLC profile of oligomers formed by mixing αA:βA3 and αB:βA3 showed a shift in elution time relative to the individual crystallins, suggesting their interaction. DLS analysis showed increase in mass from 4.11X10⁵ D (individual crystallin) up to 3.3X10⁶ D (oligomers). The MALDI analysis of cross-linked species suggested interaction of C-terminus of αB and motif III of βA3 during their interaction.

Conclusions: : Interaction between αA or αB and βA3 involves their multiple sites. Both mammalian-two hybrid and cross-linking assays revealed a greater involvement of motifs III and IV of βA3 during interaction with αA or αB crystallins.

Keywords: crystallins • gene/expression • protein structure/function