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First published online August 9, 2006
doi: 10.1242/10.1242/jcs.03061

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Brain-specific BNIP-2-homology protein Caytaxin relocalises glutaminase to neurite terminals and reduces glutamate levels

¹ Department of Biological Sciences, 14 Science Drive 4, Faculty of Science, National University of Singapore, Singapore 117543, Republic of Singapore
² Department of Anatomy, Yong Loo Lin School of Medicine, 4 Medical Drive, National University of Singapore, Singapore 117597, Republic of Singapore

View larger version (58K): [in a new window]   Fig. 1. Identification of BNIP-H and KGA interaction. (A) Bacterially expressed GST-BNIP-H immobilised on glutathione-Sepharose beads was incubated either with lysis buffer or rat brain lysate. As a control, glutathione-Sepharose beads coated with GST-BNIP-H were mixed with lysis buffer and processed the same way. After elution, bound proteins and brain lysate were resolved by SDS-PAGE and visualised by silver staining (lanes 1-5). A unique band around 70 kDa (labelled with asterisks) was subjected to trypsin-digestion followed by MALDI-TOF analysis (see Materials and Methods) and identified as kidney-type glutaminase (KGA). Comparison of eluate and brain lysate indicated an enrichment of KGA by BNIP-H (lanes 4 and 5). The identification was confirmed by western blotting with an anti-glutaminase antibody (lanes 6 and 7). (B,C) The GST pull-down and its analysis as described in A were repeated with whole-cell lysates prepared from PC12 (B) and Neuro2A cells (C). (D) The unique band, as indicated in A, was subjected to MALDI-TOF analyses as described in Materials and Methods. The tryptic peptides that correspond to and cover 27% of the protein sequence of rat KGA are underlined.

View larger version (69K): [in a new window]   Fig. 2. Characterisation of BNIP-H and KGA interaction. (A) Pre-immune serum or polyclonal antibodies against BNIP-H were used for immunoprecipitation with mouse brain lysate. The precipitate was analysed by western blotting with antibodies as indicated. Only KGA, but not an unrelated control protein (EEN/endophilin II) coimmunoprecipitated with BNIP-H. Lane 2 shows that there was no nonspecific binding to the protein A/G-beads during the pre-clearing of the brain lysate (see Materials and Methods). (B) Epitope-tagged KGA and BNIP-H expressed in 293T cells were used for co-immunoprecipitation experiments. HA-tagged BNIP-H was coimmunoprecipitated by FLAG-tagged KGA (lane 3). In the reciprocal CoIP, HA-tagged KGA could be coimmunoprecipitated by FLAG-BNIP-H (lane 4) confirming an in vivo interaction between both proteins. HA-KGA did not interact with FLAG-EF1A1 (lane 5). (C,D) Lysates from transfected 293T cells expressing the indicated proteins were immunoprecipitated with an antibody against FLAG, and bound proteins and lysates were analysed with antibodies against FLAG and HA. Schematic representations of the proteins are shown on top. (E) To show direct binding, immobilised beads of GST-BNIP-H, GST-BNIP-2 and GST-BNIP-S were mixed with a FLAG-tagged KGA fragment (aa 134-669) that was produced by in vitro transcription and translation (lane 5). After incubation and washing, the precipitated proteins were analysed by western blotting with FLAG antibody. Only GST-BNIP-H bound to the FLAG-tagged KGA fragment (lane 3), no binding was observed for GST-BNIP-2 or GST-BNIP-S (lane 1 and 2, respectively). No protein was produced in an in vitro transcription and translation reaction without template (lane 4) demonstrating the specificity of the expression system. The amido black staining shows that equal amounts of GST fusion protein were used for this assay. WCL, whole-cell lysates.

View larger version (108K): [in a new window]   Fig. 3. BNIP-H expression in different parts of the CNS. (A) BNIP-H expression was examined by western blotting with different mouse tissue lysates. A purified polyclonal antibody against human BNIP-H was used. Anti-ß-tubulin antibody was used to demonstrate equal loading. (B-E) BNIP-H expression in different parts of the CNS was examined by immunohistochemistry with mouse brain sections. Strong expression was detected in CA3 of the hippocampus (B), the cerebellar cortex (C), the deep cerebellar nuclei (D) and the pontine nuclei (E). Insets show a higher magnification of the boxed area. CA3, CA3 region of the hippocampus; C, cortex. Bars, 500 µm (B); 100 µm (C-E).

View larger version (60K): [in a new window]   Fig. 4. BNIP-H is expressed in differentiating P19 cells. (A) Lysates of untreated and retinoic acid (RA)-treated P19 cells were prepared and analysed by western blots with BNIP-H antibody (i) or glutaminase antiserum (ii). Arrow in ii indicates KGA as one of several isoforms detected in these cells by the polyclonal antiserum. The blots were then stripped and reprobed with ß-tubulin antibody to normalise for their loading. Lanes in i were derived from the same blot after removal of unrelated lanes in between. (B,C) Retinoic acid-treated P19 cells were fixed, permeabilised and probed with BNIP-H antibody, glutaminase antiserum or neurofilament-160 antibody, followed by appropriate fluorophore-conjugated secondary antibodies and analysed by confocal microscopy as described in Materials and Methods. Two sets of representative images were taken (i and ii) and each was merged (right panels) to compare their relative distribution and localisation. Bars, 10 µm.

View larger version (148K): [in a new window]   Fig. 5. BNIP-H expression as revealed by ISH histochemistry. BNIP-H expression was examined by ISH histochemistry with rat brain sections as described in the Materials and Methods. Digoxigenin-labelled riboprobe was generated with rat BNIP-H cDNA as template. Coronal sections showed strong staining of the cortex and hippocampus (A), the deep cerebellar nuclei (B), the cerebellar cortex (inset of B) and the spinal cord (C). C, cerebral cortex; ca, ventral horn; cg, central grey matter of the spinal cord; cpo, dorsal horn; GCL, granule cell layer; GD, dentate gyrus; i, interpositus nucleus; l, lateral cerebellar nucleus; m, medial cerebellar nucleus; ML, molecular layer; PCL, Purkinje cell layer. Bars, 2 mm (A,B); 100 µm (inset in B); 200 µm (C).

View larger version (126K): [in a new window]   Fig. 6. BNIP-H relocalises KGA to neurite terminals independently of mitochondria. PC12 cells were untransfected (A) or transfected with the indicated epitope-tagged expression plasmids (B-E) and stimulated with NGF for 24 hours. (A) Cells were visualised under bright field to indicate the neurite-outgrowth induced after NGF treatment. (B-E) PC12 cells overexpressing the indicated proteins were fixed, permeabilised and detected by appropriate anti-FLAG or anti-HA, followed by appropriate fluorophore-conjugated secondary antibodies and confocal microscopy detection as described in the Materials and Methods. Mitochondria were stained with MitoTracker dye. Representative images are shown either in their individual colour, merged or in bright fields. Arrows in E indicate the neurite terminals to which KGA had been relocalised by BNIP-H. However, there is no increase in the concentration of mitochondria in these sites. Bars, 20 µm (A-D); 40 µm (E).

View larger version (22K): [in a new window]   Fig. 7. BNIP-H alters the steady-state levels of glutamate and inhibits glutaminase activity. (A) 293T cells were transfected with the indicated plasmids and harvested after 24 hours. Glutamate amounts in the cell lysate (black bars) and in the medium (grey bars) were determined enzymatically as described in the Materials and Methods. The results presented are means ± s.d. of three independent transfection studies, with the measurements done in triplicate. In each experiment, KGA amounts were detected by western blotting to ensure equal expression in double-transfected cells. A representative expression analysis is shown at the bottom; signals for anti-ß-tubulin demonstrate equal loading of the whole-cell lysates. EF1A1 was used as a control and had no significant effect on the endogenous glutamate level in the cell (P=0.10) and in the medium (P=0.89). White bars show the total amount of glutamate in both the lysate and medium. Capital letters (A,B), small letters (a,b) and symbols (, ß) indicate values for intracellular, medium and total glutamate amounts, respectively. Difference between values not sharing the same letters or symbols are statistically significant at P<0.05 by analysis of variance and the Newman-Keuls multiple range test (StatsDirect). Bars showing obvious similarities in values are excluded from labelling for clarity purposes. (B) Lysate from 293T cells over-expressing FLAG-tagged KGA was incubated with equal amounts (5 µg) of bacterially expressed purified GST-tagged BNIP-H FL, GST-BNIP-H (aa 1-190) or GST. After pre-incubation, glutamine was added to the samples for 0, 2, 5 and 10 minutes. Samples were then precipitated and total amounts of glutamate were determined as in A. Results are means ± s.d. of three independent enzyme assays