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F. Rizvi, T. Heimann, W. J. O'Brien; Expression of NADPH Oxidase (NOX5) in Rabbit Corneal Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1646.
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The purpose of this study was to determine whether NOX 5 is expressed in rabbit corneal stromal fibroblast (RCSF). NADPH oxidases (NOX1-5) are enzymes that serve as electron transporter and generate of superoxide. The functions of superoxide generated by NOX1-5 include apoptosis, cell survival and growth. During the course of evolution the acquisition of calcium binding EF hand domains by NOX, led to NOX5 and DUOX like isoforms that contains an N-terminal EF-hand region and thus can be activated by calcium.
We used HuNOX5 primers to amplify cDNA made from RCSF by RT-PCR. Amplified product was sequenced to confirm the identity. The protein encoded by the NOX5 was identified by Western blot analysis. Relevant in silico analysis were performed to establish the putative binding domains, evolutionary significance and functions using web based computational tools and software such as SPLIGN, PROSITE, and CDART.
The 240 bp amplified product when sequenced and BLASTed showed 97% homology to HuNOX5 mRNA and 91% to Rabbit (Oryctolagus cuniculus) NOX5 gene fragment (BR000301). SPLIGN analysis was completed for accurate alignment of this cDNA fragment to the genomic counterparts. Alignment with HuNOX5α was 100% that helped us in identifying the inferential translation start site of NOX5 gene in rabbit. In this manner the full translated protein sequence (+1) to stop codon was obtained by using SPLIGN. The motif search, predicted the presence of at least 3 putative EF-hand motifs in the region between amino acid 57-92; 93-128 and 137-172 in N-terminal region and a FAD/NADP binding domain in C terminal region. The translated protein sequence of the cDNA fragment; identified by EMBOSS Transeq, aligned with 484 to 563 amino acid sequence of HuNOX5 region associated with Ferric Reductase /NAD Binding domain_6 and C terminal NADP linked domain. CDART analysis of fragmented 80 amino acids from rabbit linked it to pfam08030 super family, which includes cd06195 and corresponds to Ferrodoxin reducatse (FNR), a FAD and NADP binding protein. FNR has a strong preference for NADP (H) rather than NAD (H). Our assays of NADPH oxidase activity in preparations from rabbit corneal fibroblast have shown high activity with NADPH rather than NADH as a substrate. Western blots documented that a protein of about 65 kDa was present in membranes of RCSF when probed with the polyclonal antibody to NOX5.
We have identified a NOX5 homolog of NADPH oxidase in RCSF. Studies are underway to find the functional significance of oxidase and its role in O2_ production and signal transduction pathways.
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