A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells
© Weiher et al. 2016
Received: 17 July 2015
Accepted: 16 February 2016
Published: 27 February 2016
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.
Mutations in the human MPV17 gene have been firstly discovered to be causal for the lethal liver disease Mitochondrial DNA Depletion Syndrome (MDDS) by Spinazzola et al. in 2006 . Since then various additional mutations within this gene were described to cause the syndrome [5, 8, 23]. In contrast to other proteins of which gene mutations cause MDDS such as POLG, TK2, or DGOUK , this protein is not obviously involved in replication or in nucleic acid metabolism, but appears to be a membrane protein of unknown molecular function [21, 25, 26]. MPV17 gene knockout in mice has been described earlier as causal of glomerulosclerosis [2, 14, 25] and inner ear disease , reminiscent of chemical damage by Adriamycin . Although these mice also showed mitochondrial DNA depletion, they displayed no major liver phenotype . A functional link between the PRKDC repair protein, the mouse kidney phenotype as well as the mitochondrial depletion phenotype has been established by Papeta et al. . Human MPV17 protein expression can rescue the phenotype in transgenic mice, negative for murine MPV17 . A functional homolog of the MPV17 protein was identified in yeast (Sacharomyces cerevisiae) and the human gene can rescue the yeast phenotype in Sym1 negative yeast as well . In addition, a mutant in the zebra fish (Dario rerio) MPV17 homolog tra has been described . The yeast and fish mutant phenotypes appear remarkably different from the mutant phenotypes in mammals: Sym 1 negative yeast fails to grow at elevated temperature in ethanol , while tra negative fish are viable but show a transparent appearance . Finally, MPV17 is a member of a protein family, including MPV17 like proteins [6, 19] and the peroxisomal membrane protein PXMP22 [7, 18]. The hypothesis that MPV17 protein might constitute a channel allowing—in vertebrates—nucleotides or—in yeast—metabolic intermediates to pass though internal membranes was recently reviewed .
The publication describing the causative role of MPV17 mutations in the human liver disease MDDS included an analysis of the intracellular localisation of the MPV17 protein in human cells based mainly on studies of transfected cells over-expressing a c-terminally tagged recombinant protein, it was stated that this protein localised to the inner membrane of mitochondria . Supporting this notion, it has been established that the yeast homolog of MPV17, SYM1, indeed localises to this cell compartment [17, 22]. An earlier study , however, had localised the MPV17 gene product to peroxisomes, again based on immunofluorescence data and supported by the similarity of the protein to the bona-fide peroxisomal membrane protein PXMP22 .
To clarify this contradiction we here present co-localisation studies using novel anti-human MPV17 monoclonal mouse antibodies.
Cell lines and tissue culture
Human osteosarcoma cells (U2OS) cells were originally obtain from the American tissue culture collection (ATCC® HTB-96™), kept at the Heinrich Pette Institut for several years and used in . Primary murine embryonic fibroblasts (MEF) were produced from day 11 MPV17 +/+ and MPV17 −/− embryos respectively according to Zwacka et al. . U2OS and mouse MEF cells were cultured under standard conditions. Stable transformants were selected using 1 mg/ml G418 (Life Technologies). Hybridomas were generated as described , and clones were grown in RMPI media including 10–20 % FCS. The clones were monitored for IgG production by immunoblotting using anti-IgG antibodies and the ECL System (Pierce) for detection. Antibodies were purified using a Protein G Sepharose column (Life Technologies) according to the manufacturer’s instructions.
Antibodies and organelle detection
Commercial primary antibodies against MPV17 were: mouse monoclonal anti-human MPV17 antibody (60103-1-Ig, Proteintech), rabbit anti-human MPV17 polyclonal antibody (10310-1-AP, Proteintech); rabbit anti-human MPV17 polyclonal antibody (ab93374, Abcam); goat anti-human MPV17 polyclonal antibody (sc-109551, Santa Cruz), rabbit anti-MPV17 C-terminal region polyclonal antibody ARP73712-P050 Insight Biotechnology), rabbit anti-MPV17 N-terminal region polyclonal antibody AP8749a-ev-AB, BioCat).
Further primary antibodies used were: mouse polyclonal anti-human catalase antibody (Abcam ab88650); mouse monoclonal anti-complex IV mitochondrial subunit I (Invitrogen 459600); rabbit polyclonal anti-human PMP70 antibody (gift from Wilhelm Just, University of Heidelberg); rabbit anti-human Rab 7 antibody (Sigma R4779); goat anti- human cathepsin D antibody (Santa Cruz sc 6486); rabbit anti-human EEA1 antibody (Novus-Biologicals, NB-300-502); goat anti-human LAMP1 antibody (Santa Cruz, sc 8098); mouse monoclonal anti-flag antibody (Sigma, M2, F3165), mouse monoclonal anti-human beta-actin antibody (Sigma, AC74, A5316).
Primary monoclonal Antibodies 5D2 and 6F5 were isolated from mass cultures and purified over protein G Sepharose (Life Technologies). A Rabbit polyclonal anti-human PMP70 antibody was a gift from Wilhelm Just, University of Heidelberg. Further primary antibodies used were: rabbit anti-human Rab 7 antibody (Sigma R4779); goat anti-human cathepsin D antibody (Santa Cruz sc 6486); rabbit anti-human EEA1 antibody (Novus-Biologicals, NB-300-502); goat anti-human LAMP1 antibody (Santa Cruz, sc 8098); mouse monoclonal anti-human beta-actin antibody (Sigma, AC74, A5316).
Secondary antibodies: HRP labeled anti-mouse IgG antibody (Pierce); Alexa Fluor 555 and Alexa Fluor 488 with appropriate anti-mouse, anti-rabbit, and anti-goat specificity were from Life Technologies. For mitochondrial staining, MitoTracker Red 7510 was employed according to the manufacturer’s instructions.
Cell extracts and immunoblotting
An MPV17 expression construct (SC118652) was from origene. For details see: www.origene.com/cdna/.
When tested in immunofluorescence studies on U2OS cells, 6F5 did not display specific reactivity (data not shown), while 5D2 generated a characteristic punctate pattern, which in the Image J analysis of confocal microscopic pictures showed partial colocalisation with the peroxisomal marker PMP70 (polyclonal rabbit PMP70 antibody, gift from W. Just, University of Heidelberg), (Fig. 3 top). However, the pattern did not coincide with mitochondrial staining as displayed by MitoTracker Red (MP07510, Invitrogen) (Fig. 3 bottom). Thus, the staining pattern was not exclusively peroxisomal and not mitochondrial, as had been reported for MPV17 localisation in earlier studies, respectively [21, 26].
In this work we have characterised two monoclonal antibodies raised against a bacterially expressed N-terminal GST fused to the human MPV17 protein. These antibodies, specific for the MPV17 part of the fusion protein recognise a band of the expected size in human U2OS cells. While 6F5 cross reacts with other proteins 5D2 appears to be monospecific. 5D2 creates a punctate pattern in immunofluorescence studies partially colocalising with peroxisomal, early endosomal, and lysosomal markers but not with mitochondria. 5D2 does bind only weakly but specifically to the murine MPV17 homolog (Fig. 1), suggesting that the mouse monoclonal might bind to an epitope where murine and human MPV17 diverge. This could be the c-terminus of the molecule, which is the region of strongest divergence. In line with this idea is the fact that the c-terminus of the MPV17 molecule was exposed in the bacterial n-terminal fusion to GST antigen used for immunisation. Moreover, MPV17 molecules fused at the c-terminus to other proteins cannot be detected by 5D2 (data not shown).
The commercial anti-human MPV17 antibody used in our first approach was a polyclonal rabbit antibody raised against GST-MPV17 fusion protein (abcam ab 93374) and has produced erroneous or ambiguous results (Fig. 2). The rabbit polyclonal antibody from Proteintech used by  leading to mitochondrial localisation was raised against the identical GST-MPV17 fusion and might have been error—prone as well. Furthermore, in addition to possible transfection artifacts, MPV17 and related proteins such as PXMP22 may localise erroneously, when they are fused to detection tags (unpublished and .
MPV17 deficiencies in humans can cause fatal liver disease mediated by mitochondrial DNA depletion in liver cells . However, primary human liver cells were not available for our studies. We therefore had to perform this study on unrelated human tumour cells, and it is possible, that the MPV17 protein might localise differently in different cell types. Yet, preliminary studies on human primary skin fibroblasts show MPV17 colocalisation with LAMP1 as well (unpublished results) and thus corroborate the data on U2OS cells presented above. Thus, our data raise the question of how the mitochondrial MDDS phenotype is generated if the MPV17 gene product is found mainly in other organelles. It has been shown that MPV17 protein forms a membrane channel with a diameter allowing low molecular weight molecules to pass  but the role of the channel is still elusive, particularly considering the different phenotypes that mutations of it can cause in different species . We look forward to provide the antibodies described here to approach these open questions.
HW: Making of constructs, western blots, immunohistochemistry, confocal microscopy. HP and PJD: contribution to the Westernblot in Fig. 1, transfection and immunohistochemistry depicted in Fig. 2. SH and PK: help with growth and purification monoclonal antibodies. SG, MV and JKH: help with and the production of MPV17 negative MEF cells. RMZ: generation of monoclonal antibodies. ES: help with confocal microscopy and Fiji software. KR and HW: technical and financial support; critical reading of the manuscript and most valuable discussion. All authors read and approved the final manuscript.
We thank Dr. Alexander Bartelt and Dr. Katrin Kollmann, UKE Hamburg, for marker antibodies and valuable discussions. This work was supported by the Heinrich-Pette-Institute, Hamburg, Germany; the Institute for Biomedical Aging Research, University of Innsbruck, Austria; the Institutes of Molecular Medicine and Experimental Immunology, Universität Bonn, Germany; and the Department of Biochemistry, Biocenter, University of Oulu, Finland.
The authors declare that they have no competing interests.
All work concerned tissue culture cells for which no specific licences were required. For isolation of the mouse embryonic fibroblasts the use of experimental animals was conducted under the permission of the Animal Experimentation Board of Finland (ESLH-2009-04840/Ym-23).
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