Anti-FLAG tag antibody, clone O4 [AP]

Congenital cataract is leading cause of visual impairment and blindness in children worldwide. Approximately one-third of congenital cataract cases are familial, whose genetic etiology can be distinguished by targeted exome sequencing. Here, a three-generation congenital cataract pedigree was recruited, and physical and ophthalmologic examinations were taken. Targeted exome sequencing of 139 cataract-related genes was performed on the proband III:1. Sanger sequencing was used to validate the presence of variation identified via exome sequencing in family members and 200 controls. Conservative and functional prediction was performed with bioinformatic tools. We, thus, found a heterozygous missense mutation c.10T>A (p.W4R) in gap junction protein alpha 8 (GJA8) in the patients. However, this mutation was not present in normal family members and 200 unrelated controls. The GJA8 gene encodes a gap junction protein, connexin 50 (Cx50), in lens fibers that provide channels for exchange of ions and small molecules between adjacent cells. Conservative and functional prediction suggests that the W-to-R substitution at codon 4 may impair the function of the human Cx50 protein. Accordingly, we analyzed the distribution of Flag-tagged mutant Cx50 protein in HeLa cervical cancer cells. lmmunofluorescent staining showed that the W-to-R substitution impaired Cx50 trafficking to the plasma membrane to form the gap junction. In conclusion, c.10T>A (p.W4R) in GJA8 is the newly identified genetic cause of familial congenital cataract. The W-to-R substitution near the amino-terminus may alter the localization of mutant Cx50, thereby impairing gap junction formation, which is the molecular pathogenic mechanism of this mutation.

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b. The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.