Expression, Antibody Preparation And Subcellular Localization Of NRDR And Its Isoform

Retinoids are important excitatory autacoid in mammals. As the active form of retinoids,retinoic acid (RA) can be drived from retinol by two-step oxidation with retinal as intermediatein vivo. The change from retinol to retinal is the rate-limited step in this reaction. Lots ofenzymes are considered to take part in these two metabolism processes. NADP(H)-dependentretinol dehydrogenase/reductase, NRDR, was purified from rabbit liver by Huang DY. NRDRexists in mammalian livers universally, and shows high activity in retinol oxidation and retinalreduction. In addition, NRDR can catalyse the reduction of many aldehydes and ketones.The results of bioinformatics analysis show NRDR contains an N terminal mitochondrialtargeting signal (MTS) and a C terminal peroxisomal targeting sequence type 1 (PST1)containing three amino acids, Ser-Arg-Lys (SRL).Human NRDR A2, abbreviated to A2, is a novel alternative splicing isoform of NRDRdetected from human neuroblastoma cell line SK-N-SH. A2 differs from NRDR in that it lacksthe fourth and sixth exons, and the reading frame has shift forward for 1 base pair beginning withthe fifth exon. The frame shift mutation terminates protein translation in advance, and a novellocalization signal, nuclear localization signal (NLS), takes the place of PST1.Accoding to our previous experimental results and NRDR sequence analysis, it was suggestedthere are three forms of rabbit NRDR generated by alternating translational initiation site orpost-translation processing, which contain 279aa, 260aa and 256aa respectively.Functions of protein are tightly related with its localization in vivo. The changes of proteinlocalization usually result in the function alteration, accordingly. Therefore, identification thesubcellular localization of NRDR and its isoforms has great significance for the function study ofNRDR.A2 was expressed in E. coli, and the recombination protein was purified for immunizing rabbitto prepare rabbit anti human A2 antiserum. Green fluorescent protein (GFP) fusion plasmidswere constructed to demonstrate the distribution of A2 and NRDR (279/260/256) in mammaliancells. Native A2 was also expressed in mammalian cells and its subcellular location was detectedby immtmofluorescence. To study the mechanisms by which A2 is distributed, GFP fusion expression plasmids were constructed containing NLS sequence and different A2 fragmentsrespectively.Though NLS could import GFP into nuclear, A2 fused with GFP was distributed thoughout thecell, and exogenous expressed native A2 was localized in cytoplasm. Results of recombinationexpression experiment about different A2 fragments indicate that the homologous sequence withhumNRDR may impact A2 localization. All the forms of rabbit NRDR with GFP fused to their Nterminals are localized in peroxisome, and rabNRDR260 with GFP fused to its C terminal isdiffused in cytoplasm.