The terminal differentiation of lens fiber cells is one of the most characteristic and unusual features of the vertebrate eye lens. Fully mature fiber cells become tightly packed and lose their nuclei and other cellular organelles, presumably to improve clarity along the optical axis of the lens (1
). Nuclear breakdown involves condensation and degradation of chromatin in a process similar to apoptosis, following which nuclear fragments disperse. Lens cytoskeleton also undergoes reorganization during maturation (7
Lengsin is highly specific for the lens and is well conserved throughout vertebrate evolution (15
). As shown here, lengsin has a uniquely restricted pattern of expression during lens development. Expression is triggered in a specific layer of maturing lens secondary fiber cells as they approach the final stages of terminal differentiation. Lengsin mRNA is apparently tightly regulated and disappears rapidly in cells in which the nuclei have gone. A similar onset of lengsin expression is seen by immunofluorescence. Lengsin protein is seen throughout the ring of fiber cells undergoing loss of nuclei, but the protein is retained in deeper, older layers of mature fiber cells. In some sections, nuclei in the degradation zone also stained intensively for lengsin. While this could have functional significance for the mechanisms of nuclear breakdown it may simply reflect “leakiness” in the nuclei heading for condensation and breakdown that allows lengsin or antibodies to enter. A fascinating question for future studies is the nature of the signal that triggers lengsin expression in nuclei on the verge of breakdown.
The observation of lengsin as a fiber cell marker is consistent with results comparing gene expression in regenerating mouse lens (lacking mature fiber cells) with control lens. Along with several crystallins and other lens fiber markers, lengsin (incorrectly identified as glutamate-ammonia ligase, rather than glutamate-ammonia ligase (glutamine synthetase) domain containing 1), is one of the genes with the greatest decrease in mRNA levels compared with control lens (26
Despite its place in the GS superfamily, lengsin exhibits no evidence of enzyme activity (15
). Indeed, structural studies show major deletions and loss of functionally important residues in the catalytic and binding sites of lengsin compared with bacterial GS enzymes (15
). It is also difficult to imagine a GS-related enzymatic role that would explain the localized, tissue-specific expression of lengsin. Rather, lengsin may a have a structural or binding role the lens. This would have parallels with the independent recruitment of several different enzymes as novel crystallins during vertebrate evolution (27
). In these cases, enzyme activity or cofactor binding are not centrally important to the new crystallin role (although they may confer additional benefits on the lens). Indeed in some cases, notably δ
1-crystallin, derived from argininosuccinate lyase (30
), ancestral enzyme activity has been completely lost. Thus enzyme crystallins have principally structural rather than metabolic roles in the lens. Lengsin, derived from an ancient enzyme, seems to have acquired a specialized role in the lens in a similar way, although, in contrast to the major taxon-specific enzyme crystallins, lengsin is expressed throughout the vertebrates, suggesting a more ancient origin (15
). Indeed, recent genome sequencing for the Sea Urchin (Strongylocentrotus purpuratus
) has revealed the presence of multiple lengsin-like genes (15
), suggesting that the lengsin family may have had wide use in early metazoans, although in vertebrates this function has only survived in the specialized context of the lens.
When yeast two-hybrid analysis was used to search for binding partners of lengsin in adult mouse lengsin, two proteins were detected. The first was lengsin itself. Full-length lengsin bait interacted with full-length lengsin prey, consistent with the multimeric structure of lengsin (15
). The second prey for lengsin consisted of part of the helical domain of vimentin, essentially the 2B region (20
). While sequence analysis showed that other clones containing different parts of vimentin were also present in the prey library, only clones for the 2B region were detected by Y2H. The interaction between lengsin and the vimentin 2B fragment (Vim2B) was confirmed by co-expression of recombinant proteins, by co-immunoprecipitation and by SPR.
Lens also expresses two other intermediate filament proteins related to vimentin, CP49 and filensin. These proteins are highly lens-specific and are also found throughout the vertebrates, matching well with the occurrence of lengsin. The regions of these two proteins equivalent to Vim2B were also examined for any ability to interact with lengsin. In this region CP49 has some sequence identity with vimentin, while filensin is more distantly related. The 2B region of filensin expressed well in yeast but gave no evidence for interaction with lengsin in Y2H experiments. However the 2B region of CP49 consistently failed to express correctly in yeast (a truncated fusion protein was always detected) and so could not be evaluated.
As an alternative, the recombinant CP492B region was expressed as an HA-tagged recombinant protein. Using affinity chromatography it was possible to demonstrate binding of lengsin to HA-CP492B. Furthermore, SPR spectroscopy confirmed that both Vim2B and HA-CP492B bound to mouse lengsin and demonstrated similar patterns of rapid association and slow dissociation, consistent with the existence of multiple binding sites. The SPR results are also consistent with a higher affinity of lengsin for CP49 than for vimentin. These results suggest that lengsin has the ability to bind both of these important lens intermediate filament proteins, although there may be preferred interaction between lengsin and CP49, both of which are highly specialized, tissue-specific lens proteins.
Expression of lengsin in mouse lens is closely coordinated with major changes in the levels and distribution of both the major intermediate filaments of the lens cytoskeleton. Filament proteins are prone to aggregation and formation of aggregates or plaques during the reorganization or breakdown of cytoskeleton in the highly organized, protein dense environment of lens fiber lens could have serious consequences for lens transparency. One role for lengsin as a lens-specific, intermediate filament-binding protein could be to act as a “chaperone” for both vimentin and CP49 as the lens fiber cell cytoskeleton is rearranged. Lengsin could also play a role in the organization of cytoskeleton during the formation of the specialized interdigitations that lock the mature lens fiber cells together and which also form at this stage (32
). Indeed, localization of lengsin, vimentin and CP49/filensin in the region of lengsin expression in mouse lens shows that they all appear to have a principally plasmas membrane association. While signal for vimentin is relatively weak in this zone, consistent with its observed loss in maturing fiber cell, both lengsin and CP49 show strong immunofluorescence signals outlining the lens fiber cells. It has recently been shown that CP49 and filensin interact with MIP, the major membrane protein of the fiber cell plasma membrane (33
). The similar localization of lengsin and beaded filament proteins suggests that lengsin itself could be a component of the cytoskeleton in mature secondary fiber cells. Such an interaction would certainly be consistent with the tissue specificity of lengsin and the beaded filaments proteins.
It is interesting that, in the mouse lens, lengsin seems to be specific for the maturation of secondary fibers. Lengsin has not been detected in embryonic or new born mouse lens, and there is no evidence for its expression in primary lens fibers even though primary fiber cells also undergo a terminal differentiation with loss of cell nuclei (1
). This suggests that there may be different mechanisms for maturation in the two types of fiber cells. Indeed, the origins of the two populations of fiber cells are quite different. Primary fibers arise from the elongation of posterior cells in the embryonic lens vesicle and these essentially form parallel bundles of cells that fill the lens vesicle with no formation of fiber to fiber junctions or sutures. By itself, this process would produce a primitive, size-limited cellular lens. Secondary fibers are derived from anterior epithelial cells that elongate at the lens equator and wrap around the core of the lens (the so-called lens nucleus) that is formed from the primary fibers. The layers of secondary fibers meet up anteriorly and posteriorly and form sutures (4
). Secondary fiber cells may thus be the result of an elaboration of lens development to produce larger, more sophisticated lenses during the evolution of the vertebrate eye, and lengsin may have been recruited to serve as part of their cellular machinery.
Why would a GS enzyme acquire a role involving interaction with cytoskeleton? Many familiar enzymes have been shown to interact with cytoskeletal elements in the cell (34
). This may allow anchoring of enzyme complexes, enhancing the flow of metabolites by proximity rather than by free diffusion in the cytoplasm. During evolution the enzymes and the cytoskeletal proteins would be expected to co-adapt, allowing enzymes to participate in the structure and function of the cytoskeleton itself. Perhaps a class I GS acquired a role of this kind in a distant ancestor of multicellular organisms, a role that expanded in some metazoan lineages (such as that which includes Sea urchins), but in vertebrates was preserved only in the lens. While lengsin is lens-specific, its acquisition of a structural role associated with cytoskeleton illustrates more generally the evolutionary flexibility of proteins and suggests that other proteins identified with particular roles, such as enzymes, may also be playing important structural roles in cell biology.