The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity
© Cikala et al; licensee BioMed Central Ltd. 2004
Received: 16 January 2004
Accepted: 11 June 2004
Published: 11 June 2004
Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.
We have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected.
Our results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.
Apoptotic cell death plays an essential role in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss in the phospholipid asymmetry of the plasma membrane and exposure of phosphatidylserine on the outer membrane leaflet. Surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts [1, 2].
A putative receptor for phosphatidylserine binding was cloned from human and mouse macrophages . The receptor (PSR) was identified by phage display as the antigen for a monoclonal antibody generated against stimulated human macrophages. This antibody inhibited uptake of apoptotic cells by macrophages, fibroblasts and epithelial cells.
When cells that do not express the receptor and cannot engulf apoptotic cells normally, like Jurkat T, were transiently or stably transfected with PSR they gained the ability to phagocytose apoptotic cells in a phosphatidylserine specific manner. Additionally it was demonstrated that, dependent on their interaction with phosphatidylserine, phagocytosing macrophages release cytokines to suppress the inflammatory response . In further studies it was shown that the interaction of phosphatidylserine with PSR is not so much involved in tethering the apoptotic cell towards the phagocyte but rather needed for ingesting it . Anti-PSR antibodies prevented the stereospecific phosphatidylserine dependent uptake of artificial target particles. Recently it was also supposed that PSR is involved in annexin-1 mediated apoptotic cell engulfment . New work from Caenorhabditis elegans describes that embryonic cell corpses persisted for an average of 55% longer in PSR mutants when compared to wild type animals . Mice deficient in PSR showed a much stronger phenotype. They died shortly after birth due to respiratory failure and exhibited an accumulation of non-ingested dying cells in the lung together with the recruitment of inflammatory leukocytes. They also showed hyperblastic brain malformations that in some aspects were similar to such observed in caspase-9 and APAF-1 knockout mice [7–9].
A very striking feature of PSR is its enormous conservation between mammals, flies and nematodes. This initiated our interest in the question of whether this protein was also present in Hydra and if conservation of its function was preserved.
We identified a Hydra cDNA encoding PSR with high homology to the mammalian, C. elegans and Drosophila genes. Absolutely conserved between PSR from all species are three nuclear localisation signals and an AT-hook DNA binding motif. In accordance with this we found Hydra PSR localised in the nuclei of transfected Hydra cells. Mutation of one of its nuclear localisation signals blocked this localisation. We have also identified a striking structural homology of all PSRs with the enzymatic domain of FIH-1 (factor inhibiting HIF) which has a 2OG-and Fe(II)-dependent oxygenase activity . Our data suggest that the primary function of PSR in Hydra is nuclear and not directly linked to phagocytosis. This idea may also apply to mammalian PSR. Very recent work shows nuclear localisation for human PSR in a number of mammalian cell lines .
Results and Discussion
Isolation and characterisation of a Hydra homologue of PSR
We first isolated Hydra PSR using sequence information from an alignment of human and mouse PSR with the predicted ORFs from Drosophila and Caenorhabditis elegans. Degenerated primers were designed and RT-PCR was carried out with hydra mRNA as template. PCR revealed a 0.5 kb product. It contained an ORF that showed substantial homology with the published PSR sequence. We labelled the PCR product with DIG and used it as a probe to screen a cDNA library. We obtained a full-length Hydra PSR clone.
PSR shows additional sequence motifs. N-terminally to the central enzymatic domain the motif KTVRGRPKLS is found. It is completely conserved in PSR from Hydra to Homo sapiens (except an R-K exchange on position 4 in Caenorhabditis elegans) and has high similarity with an AT-hook motif. This is a short DNA binding motif first described in the high mobility group of non-histone chromosomal proteins (HMG). Since then it has been discovered in a large number of DNA binding proteins . The motif is centred on the three amino acids GRP that are necessary and sufficient to bind to DNA.
Another conserved feature in PSR from Hydra to human are several putative nuclear localisation signals. They are (141) PKKRK (145) and (167) KRRPP (171) in the predicted cytoplasmic domain and (377) KRRK (380) in the predicted extracellular domain (illustrated in Fig. 1).
In Fig. 1 we have also indicated the putative transmembrane domain that was postulated by Fadok . However, none of the transmembrane prediction programs we used (see Methods) could identify a transmembrane domain in this region or anywhere else in the hydra PSR molecule.
This prompted us to investigate the subcellular localisation of this protein. Based on the described function of human PSR in phagocytosis a membrane association was expected.
Subcellular localisation of PSR and its NLS-mutants
As described above, the PSR protein contains 3 putative nuclear localisation signals (Fig. 1). After deletion of NLS 1 or NLS 2, nuclear localisation could still be observed (Fig. 5C and 5D). Deletion of both these NLS sequences did not change the nuclear localisation of the protein either (Fig. 4D). However, deletion of the most C-terminal NLS compromised nuclear localisation of the fusion protein dramatically. This is depicted in Fig. 4E and 4F and in Fig. 5E. In GFP-PSRΔNLS3 GFP is distributed throughout the cell, both in fixed and in non-fixed hydra cells. An additional GFP fusion protein only containing the JmjC domain of PSR is localised in the same way (not shown). Interestingly, although not nuclear, these mutants are not localised to the membrane either.
From these data we concluded that Hydra PSR is normally localised in the nucleus. Nuclear localisation depends on a C-terminal nuclear localisation signal. The nuclear function of this protein has still to be established. The presence of an AT-hook in the sequence suggests an attachment to DNA. More strikingly, the putative dioxygenase activity of this molecule that is inferred by comparison of its sequence and secondary structure with FIH-1, could point to a function in the regulation of nuclear proteins by hydroxylation. This regulation would be dependent on the concentration of oxygen, as it has been described for FIH-1 and oxygen dependent activation of HIF.
Our results clearly contradict a primary function for Hydra PSR as a membrane receptor involved in phagocytosis. We suggest that it is a nuclear dioxygenase that is capable of modifying nuclear proteins. We can, however, not exclude that the protein excerts an indirect effect on apoptosis and phagocytosis. The implied oxygen dependence of its putative enzymatic activity could interact with apoptotic mechanisms. This would also provide an additional explanation for the dramatic effect on lung development observed in PSR knockout mice . In order to understand the function of PSR its intracellular targets will have to be identified.
Cloning of Hydra PSR
Degenerated primers were designed on the basis of the sequence homologies between PSR from human to Caenorhabditis elegans.
Primer 2 (forward): AARATGAARATGAARTAYTA
Primer 5 (reverse): NACNACRTGCCACCANCC
RT-PCR using hydra cDNA as a template revealed a 0.5 kb fragment. After sequencing this fragment was labelled with DIG and used to screen a Hydra cDNA library as described previously . The sequence has been submitted to GenBank (accession number AY559448).
Cloning of psr-gfp fusions in the EGFP expression vector 
First a Sma I-site was introduced into the vector 5' to the gfp-sequence by site directed mutagenesis. For PSR-GFP fusion proteins the psr sequence was introduced into this Sma1-site. For GFP-PSR fusion proteins it was cloned into the EcoR I-site. The STOP-codon 5' of this site was then removed by site-directed mutagenesis. This created a short linker between GFP and PSR encoding for the amino acids IAFVAF.
NLS-mutants were introduced by site directed mutagenesis. In ΔNLS1 amino acids 142–145 (KKRK) were deleted, in ΔNLS2 amino acids 166–168 (KRR) and in NLSΔ3 amino acids 377–380 (KRRK). The double mutant Δ1,2 was constructed by deleting amino acids 166–168 in ΔNLS 1.
DNA was introduced into hydra cells using a particle gun as described previously .
Living animals with GFP expressing cells were stained with FM-464 (Molecular Probes) for detection of plasma membranes or SYTO-15 for nuclear staining, treated with urethane and scanned immediately. Fixation was carried out with 2% paraformaldehyde in PBS for 1 h. Fixed animals were counterstained for DNA with TO-PRO-3 (Molecular Probes) and mounted on slides with VECTASHIELD mounting medium (Alexis Biochemicals).
Light optical serial sections were acquired with a Leica (Leica Microsystems, Heidelberg, Germany) TCS SP confocal laser scanning microscope equipped with an oil imersion Plan-Apochromat 100/1.4 NA objective lens. Fluorochromes were visualized with an argon laser with excitation wavelengths of 488 nm, 488 nm, 514 and emission filters 520–540 nm, 550–700 nm and 530–550 for EGFP, FM-464 and SYTO-15 respectively, and with a helium-neon laser with excitation wavelength 633 nm, emission filter 660–760 nm (for TO-PRO-3). Two fluorochromes and the phase contrast image (transmission filter) were scanned sequentially. Image resolution was 512 × 512 pixel with a pixel size ranging from 195 to 49 nm depending on the selected zoom factor. The axial distance between optical sections was 200 nm for zoom factor 4 and 1 μm for zoom factor 1. To obtain an improved signal-to-noise ratio each section image was averaged from 4 successive scans. The 8 bit greyscale single channel images were overlayed to an RGB image assigning a false colour to each channel, and then assembled into tables using Adobe Photoshop 5.5.
Apoptosis was induced with 1 μM wortmannin for 4 hours before cells were analysed. For annexin-V-fluorescin staining apoptosis was induced with 0.4% colchicine for 6 hours. Animals were then dissociated with pronase  and incubated with annexin-V-fluorescin and PI. The two fluorochromes and the phase contrast were scanned as described above.
PSORT II was used to predict the nuclear localisation signals. The same program did not predict an N-terminal signal peptide or transmembrane domain for the threshold 0.5. Furthermore for prediction of transmembrane domains we used Toppred 2  for eukaryotic cells with the default settings (Cutoff for certain transmembrane segments: 1.00, Cutoff for putative transmembrane segments: 0.60, Critical distance between 2 transmembrane segments: 2, Critical loop length: 60.) and TMHMM , also with default settings.
green fluorescent protein
open reading frame
nuclear localisation signal
hypoxia inducing factor
factor inhibiting HIF
- 2-OG 2:
This work was supported by a grant from Deutsche Forschungsgemeinschaft (DFG grant BO 1748 3-1). We also thank Stefan Paschen, Ludwig-Maximilians-University Munich, for helpful discussions.
- Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR: Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995, 182: 1545-1556. 10.1084/jem.182.5.1545.View ArticlePubMedGoogle Scholar
- Fadok VA, de Cathelineau A, Daleke DL, Henson PM, Bratton DL: Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J Biol Chem. 2001, 276: 1071-1077. 10.1074/jbc.M003649200.View ArticlePubMedGoogle Scholar
- Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM: A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature. 2000, 405: 85-90. 10.1038/35011084.View ArticlePubMedGoogle Scholar
- Hoffmann PR, deCathelineau AM, Ogden CA, Leverrier Y, Bratton DL, Daleke DL, Ridley AJ, Fadok VA, Henson PM: Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J Cell Biol. 2001, 155: 649-659. 10.1083/jcb.200108080.PubMed CentralView ArticlePubMedGoogle Scholar
- Arur S, Uche UE, Rezaul K, Fong M, Scranton V, Cowan AE, Mohler W, Han DK: Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev Cell. 2003, 4: 587-598. 10.1016/S1534-5807(03)00090-X.View ArticlePubMedGoogle Scholar
- Wang X, Wu YC, Fadok VA, Lee MC, Gengyo-Ando K, Cheng LC, Ledwich D, Hsu PK, Chen JY, Chou BK, Henson P, Mitani S, Xue D: Cell Corpse Engulfment Mediated by C. elegans Phosphatidylserine Receptor Through CED-5 and CED-12. Science. 2003, 302: 1563-1566. 10.1126/science.1087641.View ArticlePubMedGoogle Scholar
- Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H, Su MS, Rakic P, Flavell RA: Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell. 1998, 94: 325-337. 10.1016/S0092-8674(00)81476-2.View ArticlePubMedGoogle Scholar
- Li MO, Sarkisian MR, Mehal WZ, Rakic P, Flavell RA: Phosphatidylserine receptor is required for clearance of apoptotic cells. Science. 2003, 302: 1560-1563. 10.1126/science.1087621.View ArticlePubMedGoogle Scholar
- Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM, Mak TW: Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell. 1998, 94: 739-750. 10.1016/S0092-8674(00)81733-X.View ArticlePubMedGoogle Scholar
- Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK: FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev. 2002, 16: 1466-1471. 10.1101/gad.991402.PubMed CentralView ArticlePubMedGoogle Scholar
- Cui P, Qin B, Liu N, Pan G, Pei D: Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals. Exp Cell Res. 2004, 293: 154-163. 10.1016/j.yexcr.2003.09.023.View ArticlePubMedGoogle Scholar
- Dunwell JM, Culham A, Carter CE, Sosa-Aguirre CR, Goodenough PW: Evolution of functional diversity in the cupin superfamily. Trends Biochem Sci. 2001, 26: 740-746. 10.1016/S0968-0004(01)01981-8.View ArticlePubMedGoogle Scholar
- Clissold PM, Ponting CP: JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta. Trends Biochem Sci. 2001, 26: 7-9. 10.1016/S0968-0004(00)01700-X.View ArticlePubMedGoogle Scholar
- Mahon PC, Hirota K, Semenza GL: FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001, 15: 2675-2686. 10.1101/gad.924501.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee C, Kim SJ, Jeong DG, Lee SM, Ryu SE: Structure of human FIH-1 reveals a unique active site pocket and interaction sites for HIF-1 and von Hippel-Lindau. J Biol Chem. 2003, 278: 7558-7563. 10.1074/jbc.M210385200.View ArticlePubMedGoogle Scholar
- Dann C. E., 3rd, Bruick RK, Deisenhofer J: Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway. Proc Natl Acad Sci U S A. 2002, 99: 15351-15356. 10.1073/pnas.202614999.PubMed CentralView ArticlePubMedGoogle Scholar
- Rost B: PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996, 266: 525-539. 10.1016/S0076-6879(96)66033-9.View ArticlePubMedGoogle Scholar
- Aravind L, Koonin EV: The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2001, 2: RESEARCH0007.-10.1186/gb-2001-2-3-research0007.PubMed CentralView ArticlePubMedGoogle Scholar
- Aravind L, Landsman D: AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 1998, 26: 4413-4421. 10.1093/nar/26.19.4413.PubMed CentralView ArticlePubMedGoogle Scholar
- Cikala M, Wilm B, Hobmayer E, Bottger A, David CN: Identification of caspases and apoptosis in the simple metazoan hydra [In Process Citation]. Curr Biol. 1999, 9: 959-962. 10.1016/S0960-9822(99)80423-0.View ArticlePubMedGoogle Scholar
- Böttger A, Alexandrova O, Cikala M, Herold M, David CN: GFP expression in hydra. Lessons from the particle gun. Dev Genes Evol. 2002, 212: 302-305. 10.1007/s00427-002-0245-0.View ArticlePubMedGoogle Scholar
- PSORTII. [http://psort.nibb.ac.jp/form2.html]
- Toppred2. [http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html]
- TMHMM. [http://www.cbs.dtu.dk/services/TMHMM]
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