Abstract
Purpose: :
To decipher the role of the hypothetical protein FLJ33282 (FLJ) in the mouse retina, we will characterize its structure and function. FLJ was identified in a Nrl-/- retinal cDNA library yeast two-hybrid screen,1 and subsequently, in a proteomic analysis from a rod outer segment (OS) preparation.2 We hypothesize that it is a membrane protein involved in membrane transport and chaperones phototransduction proteins.
Methods: :
The mRNA and protein expression levels of FLJ in retinas from light-adapted (LA) compared to dark-adapted (DA) mice were evaluated using quantitative RT-PCR (qPCR), immunohistochemistry, immunoblot analysis (IB), and immuno-electron microscopy (EM, courtesy of Barbara Nagel). FLJ primers were used for qPCR and polyclonal rabbit antibodies were developed with predicted antigenic epitopes from amino- (FLJ-FM #76-88) and carboxy- (FLJ-LG #390-403) terminal domains.
Results: :
In situ hybridization gene expression patterns show mRNAs encoding FLJ are most abundant in the retina with prominent expression also seen in the hippocampus, cerebellum, and olfactory bulb.3 Our data confirms that FLJ is highly enriched in mouse retina and qPCR shows ubiquitous mRNA expression in retinal cDNAs with statistically significant higher levels observed in LA compared to DA. To test antibody specificity, peptide competition assays and detection of FLJ recombinant proteins were employed. IB analysis reveals that both anti-FLJ antibodies detect a 45 kDa protein in mouse retina pellet fractions but not in soluble fractions. The immunoreactive protein localizes to photoreceptor OS and outer plexiform layer (OPL). Additionally, FLJ co-localizes with Calbindin, a horizontal cell marker. Immuno-EM for FLJ shows colloidal gold particles present in the discs of photoreceptor OS with light compared to dark differences in expression.
Conclusions: :
Further experimentation is required before resolving the physiological function(s) of FLJ33282; however, based on its primary structure and subcellular location in photoreceptors and in post-synaptic OPL, we propose that this hypothetical protein may be involved in membrane protein transport in the photoreceptor inner and outer segments, as well as a role in molecular chaperoning within horizontal cell axons. Future studies are designed to test our hypothesis and further characterize the role of this novel hypothetical protein in the retina.1 Zuniga FI et al. IOVS 2007; 48: ARVO E-Abst.6012 Kwok MC et al. Mol Cell Prot. 2008; 7:1053-10663 St. Jude’s Brain Gene Expression Map (www.stjudebgem.org )
Keywords: photoreceptors • synapse • signal transduction