title: Cystathionine beta synthase expression in mouse retina.
abstract: Abstract Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. 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.
  
  
  METHODS
  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 the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells.
  
  
  RESULTS
  RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs(-)(/)(-) mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S.
  
  
  CONCLUSIONS
  We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.
authors.affiliation: Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912-2000, USA.
authors.forename: Shanu
authors.initials: S
authors.lastname: Markand
authors.forename: Amany
authors.initials: A
authors.lastname: Tawfik
authors.forename: Yonju
authors.initials: Y
authors.lastname: Ha
authors.forename: Jaya
authors.initials: J
authors.lastname: Gnana-Prakasam
authors.forename: Srinivas
authors.initials: S
authors.lastname: Sonne
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: Nilkantha
authors.initials: N
authors.lastname: Sen
authors.forename: Ming
authors.initials: M
authors.lastname: Xian
authors.forename: Sylvia B
authors.initials: SB
authors.lastname: Smith
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000502:physiology; D005978:Glutathione / Q000378:metabolism; D006712:Homocystinuria / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D061251:Primary Cell Culture; D012160:Retina / Q000378:metabolism / Q000503:physiopathology*; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000201:enzymology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000201:enzymology; D013045:Species Specificity
chemical_list: D000975:Antioxidants; D003541:Cystathionine beta-Synthase; D005978:Glutathione