Several models of inherited cataract in mice have been created by chemical or radiation mutagenesis [3
]. Many of these are associated with single base changes or small insertion/deletions that modify the coding sequence of one of the crystallins, the major soluble proteins of the eye lens. No3
is a nuclear cataract observed in a chemical mutagenesis study. The causative mutation was localized to the region of mouse chromosome 1 that contains the genes for βA2-crystallin and the cluster of six genes for the γA-F-crystallins. Since mutations in murine γ-crystallin genes often produce severe phenotypes, this raised the interesting possibility that No3
could be the first example of a mutation in βA2-crystallin. However, this proved not to be the case and, surprisingly, considering the reported severity of similar mutations [6
was found to be associated with a fourth motif truncation mutation of γE-crystallin. In the generally similar Elo
truncation mutants, lens fiber cell necrosis and microophthalmia have been reported [6
], while the No3/No3
lens appears superficially normal in organization and only exhibits a nuclear opacity. Furthermore, the severity of this opacity is variable and strongly strain-dependent.
It was also surprising to find that the cause of the mutation was a probably spontaneous insertion of a full-length murine endogenous retrovirus, rather than chemical mutagenesis. Endogenous retroviruses (ERV) make up a large fraction of the mammalian genome [24
]. While the majority of the ERV elements are incomplete and apparently inactive, some full-length copies exist and may give rise to daughter elements that can insert elsewhere in the genome. In mice, the major class of actively transposing ERV elements is known as intracisternal A particles (IAP), and those with long terminal repeats also carry the designation for LTR. The frequency of IAP/ERV insertion varies with mouse strain; the most susceptible strain being C3H/He [24
]. The 5kbp insertion in the Cryge
gene in No3
belongs to the ERV-K class of IAPLTR1 transposons [24
], with a perfectly conserved 354bp LTR at both ends and a characteristic direct repeat (GGCGGC) at the insertion site. Genotyping confirmed that this insertion is unique to No3
and was not present in parental strains.
The ERV insertion into exon 3 of Cryge
disrupts the coding sequence of γE-crystallin approximately half way through the fourth and final “Greek-key” like motif of the protein at codon 142, after the first β-strand of the motif but eliminating the final three β-strands needed to complete the structure of the C-terminal domain [20
] (). The open reading frame continues into the ERV LTR sequence for a further 14 codons. Thus the γE polypeptide in No3
is longer than the truncated mutants of Elo
(which are also fourth motif truncations of γE) and is potentially able to form an additional β-strand. The No3
mutant protein would not be able fold correctly and would be expected to be entirely insoluble. However, since the severity of the No3
cataract was so different from that reported for Elo
, we considered the remote possibility that the apparent differences in the effects of the mutant proteins might be due to altered folding, or even formation of multimers, that might permit at least partial solubility for No3.
To test the solubility issue, recombinant γE-No3 was expressed in E.coli. The protein expressed well, but, in contrast to wild type FVBN γE, was undetectable in the soluble fraction. Attempts were made to denature and refold the insoluble protein but no solubilization was achieved. This suggests that the No3 mutant γE is indeed completely insoluble and no more functional in the lens than its counterparts in the other cataract models. There is still the possibility that the mild phenotype in No3 is due to a difference in the aggregation properties of the mutant γE (perhaps a reduced tendency for amyloid formation). However other more likely explanations for the mild phenotype are available.
Quantitation of transcript levels in No3
shows that expression of the mutant Cryge
gene is suppressed. Retroposons, particularly those with LTR sequences can suppress expression of nearby genes, either transcriptionally or post-transcriptionally [24
]. Quantitative RT-PCR of crystallin gene transcripts from +/+ and No3/No3
littermates showed that levels of mRNA for γA and γD were similar but that the combined level of mRNA for the highly similar γE and γF-crystallins (indistinguishable in this assay) was significantly reduced. Since Crygf
(the γF gene) is normal in sequence and is also the most distantly located member of the γ-crystallin gene cluster from the mutated Cryge
(), it is unlikely that its level of mRNA is significantly affected. This suggests that the level of mRNA for γE is very low in the No3/No3
lens. This may act to offset the severity of the No3
mutation at the protein level by reducing the amount of insoluble, aggregated protein in the lens fiber cells.
Opacity due to No3
may be the result of insolubilization of the mutant protein and/or to loss-of-function of γE-crystallin. Indeed, a possible functional interaction for γE-crystallin with the major lens membrane protein MIP/APQ0 has recently been reported [27
mice may be close to a “knock out” or “knock down” of γE that could be used to further investigate its role and to shed some light on the functional basis for the loss of γE expression as part of a suite of molecular changes in the evolution of the human lens [28
The severity of cataract associated with the No3 mutation is also affected by differences in genetic background. This is illustrated by the incomplete penetrance of No3 in C57BL/6 matings, which suggests that another genetic variant in the C3H/HeH strain is required for formation of an obvious opacity. In other words the No3 mutation alone is not sufficient for severe opacification of the lens but combines with some modifier that itself does not produce cataract in isolation. For example, it is possible that a coding sequence polymorphism exists in C3H/HeH that compromises the solubility or function of another lens protein but is ameliorated by normal levels of wild type γE, perhaps by direct protein-protein interaction. Loss of γE, whether by insolubilization or suppression, might then lead to loss of function or increased insolubilization of the partner protein, increasing the severity of opacity due to mutant γE alone. In strains that lack the modifier, such as C57Bl/6, the effects of the No3 mutation on γE would have a milder phenotype than in the C3H/HeH parental strain.
One possible modifier for lens phenotypes has recently been observed in mice of the 129 strain families. This involves a truncation and loss of the major lens cytoskeleton protein Bfsp2/CP49 [30
mice on the original background were genotyped for the known CP49 mutation but only wild type alleles were observed, suggesting that at least one other genetic variant capable of modifying lens function or severity of cataract exists in laboratory mouse strains and awaits discovery.
Many simple mutations in crystallins have been associated with severe inherited cataract in both humans and mice. However, these results show that in some cases the crystallin mutation may be just the final step in a multifactorial process, with the phenotype and severity of the cataract dependent also on genetic background and environment. Such a multi-step progression towards cataract may be similar to what happens in human age related cataract, as accumulating post-translational modifications in the aging lens interact with polymorphic variant sequences, eventually producing opacity that is dependent upon both genetic and lifestyle differences.