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Shanu Markand, Amany M. Tawfik, Yonju Ha, Jaya Gnana-Prakasam, Cory Williams, Srinivas Sonne, Vadivel Ganapathy, Sylvia B. Smith; Cystathionine Beta Synthase (cbs) Expression In Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3966.
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CBS is a key enzyme in the transsulfuration metabolic pathway that converts homocysteine to cystathionine, which is further converted to cysteine required for synthesis of glutathione (GSH), a major antioxidant in retina. The transsulfuration pathway has been characterized in liver, kidney, CNS, small intestine and pancreas; however studies in the eye have been limited. Enzyme activity assays suggest that CBS is present in human and pig retina, however recent immunohistochemical studies reported that CBS is not present in mouse retina (Mikami et al, 2011). We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level.
Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for analysis of: (1) cbs gene expression by qRT-PCR using primers specific for mouse cbs, and (2) CBS protein expression by Western blotting using rabbit polyclonal anti-CBS antibody. Eyes were harvested from these mice and used for immunodetection of CBS in retina. Three retinal cell types (ganglion, Müller and RPE) were isolated from WT mice for immunocytochemical analysis of CBS.
qRT-PCR revealed robust cbs expression in WT liver, and expression also, albeit much lower, in WT retina. There was no cbs expression in cbs-/- mouse retina (or liver). Western blotting detected a band of the expected size (~60 kD) in retina and liver of WT mice, but not cbs-/- mice. In immunohistochemical studies of the intact retina, CBS was present most abundantly in the ganglion cell layer of WT retina. It was detected also in primary isolations of WT Müller, RPE and ganglion cells.
We have compelling molecular evidence that CBS is indeed expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in generation of GSH in mouse retina.
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