Effects of truncations in the N‐ and C‐terminal domains of filensin on filament formation with phakinin in cell‐free conditions and cultured cells

Filensin and phakinin are lens fiber cell‐specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N‐ and C‐terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30–416) and Fil(30–369), respectively, were examined. Fil(30–416) lacks the N‐terminal 29 amino acids and the C‐terminal 248 amino acids. Fil(30–369) lacks the N‐terminal 29 residues and the C‐terminal 295 residues. In cell‐free assembly characterized by electron microscopy, filensin and Fil(30–416) co‐polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30–369) formed only aggregates. In cultured SW‐13 and MCF‐7 cells expressing fluorescent fusion proteins, filensin and Fil(30–416) co‐polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30–369) gave aggregates. Therefore, while truncation of the N‐terminal 29 amino acids did not affect filament formation, truncation of the C‐terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370–416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification.