Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.

The ocular lens is a transparent tissue composed of highly differentiated and elongated lens fiber cells that synthesize lens-specific proteins. Filensin (BFSP1) and CP49 (phakinin, BFSP2) are two lens fiber cell–specific proteins that co-assemble to form a unique intermediate filament structure: the lens beaded filaments (BFs).1–4 Primary amino acid sequence analysis shows that filensin and CP49 are homologous to other members of the intermediate filament protein family.2,3,5–7 The structure of filensin consists of a head domain, a rod domain that can be divided into three subdomains (1A, 1B, and 2), and a C-terminal tail domain,8 whereas CP49 completely lacks the non-α-helical C-terminal domain.5 The lens beaded filament has a 6- to 8-nm filament backbone that is decorated by 12 to 15 nm beads at regular intervals. This structure is found exclusively in the fiber cells of the lens and is believed to play a functional role that is unique to lens fiber cell biology. Studies have shown that knockout of filensin or CP49 each reduces the expression of the other co-assembly partner and causes earlier emergence of opacification.9,10 Structural changes in fiber cell shape and plasma membrane organization were also observed in a CP49 knockout model.11 Some forms of hereditary cataracts in humans have been linked to mutations of filensin or CP49.12–14 These data suggest that the lens beaded filaments play an important role in the development and maintenance of lens transparency. Posttranslational modifications increase functional diversity of proteins, adding an ever-increasing complexity to the functions of the cell. In long-lived lens fiber cells, both regulatory and age-related modifications can be found. Both filensin and CP49 are reported to be phosphorylated proteins15,16; however, no phosphorylation sites on filensin and CP49 have been identified. Ser 5 of filensin located in a conserved di-arginine/aromatic residue motif (YRRSY), a consensus site for protein kinase A phosphorylation14; however, phosphorylation on Ser 5 in vivo has not been reported. Both filensin and CP49 have been shown to be extensively processed during lens fiber cell differentiation.17–19 At least two major filensin fragments are formed during proteolytic cleavage—one containing the N-terminal domain and the other containing the C-terminal non-α-helical tail domain17—and a change in cellular localization of these two fragments during fiber cell differentiation was reported.17,20 Both fragments have similar electrophoretic mobilities (51–54 kDa) by SDS-PAGE.2,17,19 One of the cleavage sites was estimated to be in the vicinity of residue 453, by measuring the molecular mass of the truncated fragment18; however, detailed information about the cleavage site(s) is missing. It is important to map the proteolytic pathway of filensin and CP49 to understand the normal and pathogenic roles of these proteins. In this article, the nearly complete sequences of filensin and CP49 were mapped and sites of truncation and phosphorylation were identified. Two unexpected posttranslational modifications including postproteolytic N-acetylation and N-myristoylation were characterized. This study represents the first comprehensive examination of posttranslational modifications of lens fiber cell–specific intermediate filament proteins. The newly identified postproteolytic N-acetylation and N-myristoylation events provide insight into the functional consequence of filensin degradation during lens fiber cell differentiation.