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Shylaja Hegde, Kiran Srivastava, Om P Srivastava; Determination of Function of βA3/A1-Crystallin in Lens Development using βA3/A1-Crystallin Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5727.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of the study was to determine the function of βA3/A1 crystallin in lens development.
The βA3/A1 knockout (KO) mouse was constructed by floxing exon 4 of βA3/A1 gene. To specifically knockout the expression of βA3/A1 in the lens, the βA3/A1 KO flip positive mice will be crossed with lens-specific cre MLR-10 mice (βA3/A1 expression KO only in lens epithelium). Lenses from βA3/A1 KO- and age matched WT mice lenses were analyzed by SDS-PAGE, Mass spectrometry, western blots, qRT-PCR and immunohistochemistry.
Phenotypically βA3/A1 heterozygous KO lens did not exhibit any lens abnormality compared to WT littermates. A comparison of protein profile by SDS-PAGE showed an increased expression of ~240 kDa band and a loss of ~35 kDa band in βA3/A1 heterozygous KO lenses. Mass spectrometric analysis identified 240 kDa band as α-spectrin protein. Immunohistochemical analysis of lenses of 4-weeks old βA3/A1 heterozygous KO mice with WGA-TRITC labelling showed thinner fiber cell membrane compared to the age matched WT fiber cell membrane.
α-Spectrin plays an important role in stabilizing membrane structures, cell shape and in linking the cytoskeleton to plasma membrane or intracellular vesicles. During terminal differentiation of the lens fiber cells, the ~ 240 kDa α-spectrin is cleaved by a caspase to yield a ~150 kDa N-terminal product and C-terminal products of ~120 kDa and ~35 kDa. However, the cleavage of full-length α-spectrin was reduced in βA3/A1 heterozygous KO lenses. We speculate that the reduced cleavage of α-spectrin, in turn affects remodeling and stabilization of the fiber cell membrane in βA3/A1 heterozygous KO lenses.
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