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Comparative Study
. 2004 Jun 11:5:26.
doi: 10.1186/1471-2121-5-26.

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity

Mihai Cikala  1 , Olga Alexandrova, Charles N David, Matthias Pröschel, Beate Stiening, Patrick Cramer, Angelika Böttger
Affiliations
Comparative Study

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity

Mihai Cikala et al. BMC Cell Biol. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
Hydra PSR sequence Clustal alignment of PSR sequences from Hydra (H. vul, accession number AY559448), Caenorhabditis elegans (C. ele, NP500606), Drosophila (D. mel, NP651026), mouse (M. mus, AK122317.1) and human (H. sap, BAA25511). The JmjC domain is labelled with a red box, AT-Hook with a blue box. The putative nuclear localisation signals are depicted in green. A black dotted box encircles the previously postulated transmembrane domain [3].
Figure 2
Figure 2
Conserved hydrophobic residues between human FIH-1 and Hydra-PSR Ribbon diagram of the conserved core domain (amino acids W179 to K298) of the human FIH-1 structure (structure from [15] using RIBBON (Carson 1997). The active site residues H199, D201 and H272 in FIH are highlighted in blue. The hydrophobic residues that are conserved between Hydra-PSR and human FIH-1 are highlighted in magenta.
Figure 3
Figure 3
Alignment of the conserved core of human FIH-1 with the homologous region from Hydra-PSR Alignment of the conserved core of human FIH-1 forming the central β-barrel structure that harbours the active site with the homologous region from Hydra-PSR. Extended β-strands are depicted by blue arrows, for Hydra-PSR above the sequence, for human FIH underneath and numbered according to the published crystal structure [15]. Yellow boxes indicate the residues necessary to complex the active site Fe2+ ion, violet box indicates the lysine implicated in 2-oxo-glutarate binding. The hydrophobic residues that stabilise the barrel structure are highlighted in grey. Stars indicate residues within 4 Angstrom of the catalytic H-X-D that constitute the active site.
Figure 4
Figure 4
PSR localisation in living animals Single optical sections of GFP expressing cells of living hydra after transfection with GFP-PSR (A and B), PSR-GFP (C), NLS-mutants ΔNLS1-2 (D) and ΔNLS3 (E and F). Left hand panels show GFP, middle panels nuclear staining with SYTO-15 or membrane staining with FM-464 as indicated. Right hand panels are merged images. V indicates vacuoles typically seen in living hydra cells.
Figure 5
Figure 5
PSR localisation in fixed animals Single optical sections of GFP-PSR (A), PSR-GFP (B) and single NLS-mutants ΔNLS 1, 2 and 3 in single GFP expressing cells after fixation (C-E). Left hand panels detect GFP, middle panels DNA-staining with TO-PRO and right hand panels represent merged images. Nuclear morphology is typical for hydra epithelial cells after fixation. Note that nucleoli are not stained with TO-PRO (middle panel) and also free of GFP (left hand panel).
Figure 6
Figure 6
Phosphatidylserine on the extracellular face of apoptotic hydra cells (A) annexin-V-fluorescin label of a hydra cell after induction of apoptosis with colchicine, (B) phase contrast image of the cell, (C) absence of propidium iodid (PI) staining: PI can only enter cells after membrane integrity is lost, absence of PI staining indicates that annexin-V-fluorescin binds the extracellular face of the membrane.
Figure 7
Figure 7
PSR stays in the nucleus of phagocytising cells Hydra epithelial cell expressing GFP-PSR in the nucleus. The cell has engulfed two apoptotic cells with pycnotic nuclei (red circles). Two additional apoptotic cells are still on the outside of the cell surrounded by its membrane (red circles). They are apparently in the process of being phagocytised. Apoptosis was induced with wortmannin. Single sections of confocal images are shown: (A) phase contrast, (B) merged image from (C) and (D), (C) TO-PRO-DNA staining, (D) GFP. In (A) a green line marks the outline of the transfected cell. Yellow arrow indicates the gold particle. Scale bar 10 μm.
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