Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin

The aim of this study was to determine relative importance of N-terminal domain and C-terminal extension of alpha A-crystallin during their in vitro complex formation with phakinin and filensin (the two lens-specific intermediate filament [IF] proteins). Cloned phakinin, filensin and vimentin were purified under a denaturing conditions by consecutive DEAE-cellulose-, hydroxyapatite- and Sephadex G-75 column chromatographic methods. WT alpha A-crystallin, alpha A-NT (N-terminal domain [residue number 1 63])-deleted and alpha A-CT (C-terminal terminal extension [residue number 140-173]-deleted), were cloned in pET 100 TOPO vector, expressed in BL-21 (DE3) cells using 1% IPTG, and purified using a Ni2+-affinity column. The following two in vitro methods were used to determine complex formation of WT alpha A, alpha A-NT, or alpha A-CT with phakinin, filensin or both phakinin plus filensin together: an ultracentrifugation sedimentation (centrifugation at 80,000 x g for 30 min at 20 degrees C) followed by SDS-PAGE analysis, and an electron microscopic analysis. In the first method, the individual control proteins (WT-alpha A, alpha A-NT and alpha A-CT crystallin species) remained in the supernatant fractions whereas phakinin, filensin, and vimentin were recovered in the pellet fractions. On complex formation by individual WT-alpha A-, alpha A-NT or alpha A-CT-species with filensin, phakinin or both phakinin and filensin, WT-alpha A and alpha A-CT were recovered in the pellet fraction with phakinin, filensin or both filensin and phakinin, whereas alpha A-NT remained mostly in the supernatant, suggesting its poor complex formation property. EM-studies showed filamentous structure formation between WT-alpha A and aA-CT with phakinin or filensin, or with both filensin and phakinin together but relatively poor filamentous structures with alpha A-NT. Together, the results suggest that the N-terminal domain of alpha A-crystallin is required during in vitro complex formation with filensin and phakinin. (C) 2017 Published by Elsevier Inc.