Philipp Rovina

Markus Jaritz

Siegfried Höfinger

Christine Graf

Piroska Dévay

Andreas Billich

Thomas Baumruker

Frédéric Bornancin
email: frederic.bornancin@novartis.com

Frédéric Bornancin٭

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Ceramide kinase (CerK) was identified recently by Sugiura et al. [START_REF] Sugiura | Ceramide kinase, a novel lipid kinase[END_REF]. It is so far the only established enzyme that uses ceramide as a substrate to produce ceramide-1-phosphate (C1P). A ceramide kinase-like protein as well as a multi-lipid substrate kinase were also identified as close homologs within the diacylglycerol kinase superfamily, but their activity towards ceramide is either lacking [START_REF] Tuson | Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26)[END_REF][START_REF] Bornancin | Characterization of a ceramide kinase-like protein[END_REF] or controversial [START_REF] Waggoner | MuLK, a eukaryotic multi-substrate lipid kinase[END_REF][START_REF] Van Overloop | Further characterization of human ceramide kinase[END_REF]. C1P was first described in brain synaptic vesicles, HL60 cells and neutrophils [START_REF] Bajjalieh | Synaptic vesicle ceramide kinase[END_REF][START_REF] Hinkovska-Galcheva | The formation of ceramide-1-phosphate during neutrophil phagocytosis and its role in liposome fusion[END_REF][START_REF] Kolesnick | Characterization of a ceramide kinase activity from human leukemia (HL60) cells[END_REF], and there is now evidence that this lipid is endowed with signaling functions. Chalfant and coworkers showed that C1P participates in eicosanoid synthesis [START_REF] Pettus | Ceramide kinase mediates cytokine-and calcium ionophore-induced arachidonic acid release[END_REF], where it synergizes with sphingosine-1-phosphate (S1P) [START_REF] Pettus | The coordination of prostaglandin E2 production by sphingosine-1-phosphate and ceramide-1-phosphate[END_REF]. Their work identified the first molecular target of C1P, namely the cytosolic phospholipase A2 alpha (PLA2GIVA) [START_REF] Pettus | Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2[END_REF]. Other studies have pointed to a role of ceramide kinase/C1P in mast cell degranulation [START_REF] Mitsutake | Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells[END_REF], regulation of acidic sphingomyelinase and control of apoptosis and survival in bone marrow derived macrophages [START_REF] Gomez-Munoz | Ceramide-1phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages[END_REF][START_REF] Gomez-Munoz | Ceramide-1-phosphate promotes cell survival through activation of the phosphatidylinositol 3-kinase/protein kinase B pathway[END_REF], calcium mobilization in GH4C1 pituitary and Jurkat T-cells [START_REF] Tornquist | Ceramide 1phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells[END_REF][START_REF] Colina | Ceramide-1-phosphate induces Ca 2+ mobilization in Jurkat T-cells by elevation of Ins(1,4,5)-P3 and activation of a store-operated calcium channel[END_REF], and dopamine release in PC12 cells [START_REF] Jeon | Dopamine release in PC12 cells is mediated by Ca 2+ -dependent production of ceramide via sphingomyelin pathway[END_REF]. A role of C1P in fibroblast proliferation was proposed a decade ago [START_REF] Gomez-Munoz | Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides[END_REF][START_REF] Gomez-Munoz | Stimulation of DNA synthesis by natural ceramide 1-phosphate[END_REF]. However, this may not be general as such an effect was not observed in FRTL-5 thyroid cells [START_REF] Hogback | Ceramide 1-phosphate increases intracellular free calcium concentrations in thyroid FRTL-5 cells[END_REF].

In contrast to C1P signaling, characterization of CerK at the molecular level has been poorly addressed. In particular, there is no detailed information regarding what determines the ability of CerK to associate with the Golgi complex and plasma membrane [START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. We therefore performed a modeling and comprehensive mutagenesis analysis of the N-terminal Pleckstrin Homology (PH) domain of CerK. We identified an unusual loop interconnecting the β6 and β7 strands of this domain, that provides key positively charged residues required for localization and function of this enzyme.

Enabled/VASP homology domain-1 [START_REF] Prehoda | Structure of the enabled/VASP homology 1 domain-peptide complex: a key component in the spatial control of actin assembly[END_REF] and the phosphotyrosine-binding (PTB) domain [START_REF] Zhou | Structure and ligand recognition of the phosphotyrosine binding domain of Shc[END_REF] ranked very low (average rank < 40). To find the best fitting structural template for the human CerK PH domain, we inspected the sequence-structure alignments manually. The final model was created with MODELER (Accelrys) [START_REF] Marti-Renom | Comparative protein structure modeling of genes and genomes[END_REF], based on the sequence-structure alignment.

Electrostatic Potential (ESP) Calculations -ESP calculations based on solutions to the

Poisson Boltzmann (PB) equation were carried out with the help of program POLCH [START_REF] Hoefinger | Solving the Poisson-Boltzmann equation with the specialized computer chip MD-GRAPE-2[END_REF].

Assuming aqueous solution the dielectric constants were set to 80.0 (bulk) and 4.0 (protein interior). The obtained model structure was stored in pdb format and processed with the program XLEAP from the AMBER suite of programs [START_REF] Cornell | A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules[END_REF]. Default AMBER RESP charges as well as default AMBER van der Waals radii have been assigned (version 6.0). The wt model structure showed a net charge of +7 prior to PB calculation. For generation of an appropriate molecular surface the program MSROLL was used (version 3.8) employing a probe sphere radius of 2.0 Ǻ and resolution/fineness parameters of -f 1.0 and -l 0.3 [START_REF] Connolly | The molecular surface package[END_REF]. Resulting solvent accessible surfaces (SASA) were composed of approximately 15,000 surface elements (of flat triangular shape). Upon termination of the PB calculation the structures were embedded in cubic grid boxes of dimension 70 x 70 x 70 Ǻ with an edge length of roughly 130 Ǻ. The ESP was computed at all the grid points including the PB-obtained set of apparent surface charges distributed over the SASA. Grid points approaching any protein atom closer than 3 Ǻ were excluded from ESP calculation. Grid maps were superimposed on the protein structure and visualized with VMD [32]. Representation of the ESP was done with a color coded iso-surface mode. Selected isolevels were +/-144 mV (blue/red).

Human CerK constructs and transfection -CerK cDNA, corresponding to Genebank TM accession number AB079066, was obtained and subcloned in Gateway TM compatible entry vectors as described previously [START_REF] Billich | Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases[END_REF][START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. Site directed mutagenesis was performed using the following primers (only the forward primers are indicated, changed bases are underlined).

Combinations of mutants were performed using the same primers on already established mutants. All constructs were transferred to pcDNA6.2DEST in order to express untagged proteins or pcDNA-DEST53 in order to express N-terminal GFP-fusion constructs. For transfection, COS-1 (obtained from DSMZ and cultured in DMEM/ 10 % FCS at 37°C/5% CO 2 in a humidified atmosphere) were seeded at 10 5 cells /well in 6-well plates. After 24 hr, cells were transfected with 3 µg vectors containing expression constructs for various CerK alleles, using FuGENE 6 (Roche Molecular Biochemicals). Cells were incubated for 24 to 48 hr following transfection before harvesting.

Fluorescence microscopy -COS-1 cells were seeded into 4-chambered coverslips (LabTEK TM II -Nalge Nunc) at 2.2x10 4 cells per chamber. 24 hr later cells were transfected with 0.6 µg plasmid per chamber using FuGENE 6. Live-cell fluorescence microscopy was done 24 hr after transfection on an inverted microscope Axiovert 200 M equipped with a high resolution microscopy camera AxioCam MRc (Zeiss) and objectives Plan-Neofluar 40x/1.30 oil DIC and Plan-Apochromat 63x/1.40 oil DIC, using the following filter set: Ex 470/Em 525 nM for GFP fluorescence and 546/590 nM for red fluorescence. For quantification of regions of interest (Fig. 4A), pictures were analysed using the AxioVision 4.0 V 4.3.0.101 (Zeiss) software. The first region of interest (x) was defined at the Golgi. A second region of interest was defined on the opposite side of the nucleus (y). Background fluorescence was measured in the extracellular space (z). Golgi/cytoplasm fluorescence ratio values were calculated using the following equation: (x-z)/(y-z).

Cellular fractionation -For Fig. 1C, a one-step fractionation protocol was used. Cells were harvested at 4°C for 2 hr on a plate shaker set at maximal speed, in 20 mM Tris pH 7.5, 1 mM DTT, with protease inhibitors, and 2 % Triton X-114, 2 mM EGTA, 500 mM KCl, the latter three either alone or in combination. Cell lysates were spun down at 55,000 rpm for 30 min at 4°C in a TLA120.2 rotor using a TL-100 Ultracentrifuge (Beckman). We did not make use of clouding properties of Triton X-114 (Triton X-100 can also be used, giving rise to similar results).

For Fig. 4B, fractionation was performed in 3 steps. Cells were first scraped and homogenized by 20 strokes in a Potter-Elvejhem homogenizer, with 500 µl of buffer 1 (20 mM Tris pH 7.5; 1 mM DTT; protease inhibitors). Lysates were centrifuged at 55,000 rpm in a TLA120.2 rotor (Beckman), yielding a cytosolic fraction. Pellets were dissolved in 500 µl of buffer 2 (20 mM Tris pH 7.5; 1 mM DTT; 250 mM NaCl; protease inhibitors) and samples spun as before.

Perimembrane fractions were stored and pellets dissolved in 500 µl of buffer 3 (20 mM Tris pH 7.5; 1 mM DTT; 1 % Triton X-100; protease inhibitors). Samples were spun again and supernatants collected (Triton extract). Final unextracted pellets were resuspended in 500 µl of buffer 1 and stored.

Protein expression and purification -CerK-FLAG proteins were obtained following immunoprecipitation. 48 hr after transfection, cells were washed once with PBS and then lysed with 1 ml of lysis buffer (provided with the Sigma FLAG ® -Immunoprecipitation kit), completed with protease inhibitors. Samples were incubated for 2 hr at 4°C with vigorous shaking, spun down at 4°C for 30 min at 14,000 rpm in an Eppendorf 5402 Thermo Centrifuge. 40 µl of Anti FLAG M2 affinity gel (beads) were washed four times with the provided wash buffer, and added to each obtained supernatant. Samples were tumbled at 4°C on a wheel overnight. Subsequently, the beads were collected after a short spin and washed three times with wash buffer. Elution was performed by tumbling at 4°C for 2 hr in 300 µl of wash buffer containing 300 ng/µl of a provided 3xFLAG peptide solution. Expression plasmids for GST tagged proteins used as controls for the protein-lipid overlay experiments were as follow: murine GRP1-PH, rat PLCδ1-PH, human Hrs1-FYVE domain, all in pGEX-2TK and human DAPP1-PH, human dynamin-1-PH in pGSTag. Plasmids were transformed into TOP10 chemically competent E. coli cells. A single colony was grown over night in liquid culture. The culture was then diluted to reach an OD 600 of 0.06 and grown further until an OD 600 of 0.6. Protein expression was induced with isopropyl-beta-D-thiogalactopyranoside (500 µM) and the suspension was shaken (200 rpm) for 2 hr at 30°C. Finally cells were spun down and pellets resuspended in 1ml of lysis buffer (50 mM Tris/HCl pH 7,5; 270 mM sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM THP, 1x Phosphatase Inhibitor cocktail, 1x Protease Inhibitor cocktail, 1% Triton X-100). Afterwards samples were snap frozen in liquid nitrogen and thawed. 100 ng/µl lysozyme were added followed by a 10 min incubation on ice. Samples were sonicated for 30 s on ice using a BANDELIN Sonopuls HD2070 (set at 20%). Lysates were spun down for 30 min at 14,000 rpm, 4°C, in an EPPENDORF 5402 Thermo Centrifuge. Glutathione Sepharose ® 4B -80 µl per samplewas washed 3 times in 0.5 ml of lysis buffer. Supernatants were applied to sepharose beads and tumbled for 1 hr at 4°C on a wheel. After a short centrifugation, supernatants were discarded and the sepharose beads washed 3 times with lysis buffer supplemented with 500 mM NaCl. This was followed by ten washing steps with 300µl wash buffer (lysis buffer without EDTA and without Triton X-100). Elution was performed on a wheel in 500µl of elution buffer (wash buffer + 20 mM reduced glutathione) for 15 min at room temperature. Eluates were made to 30 % glycerol and snap frozen until use. In vitro translation of untagged CerK was performed using the TNT coupled reticulocyte lysate system (Promega) as already described [START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. Briefly 1µg of pcDNA 6.2 containing a CerK WT or mutagenized cDNA sequence was mixed with 25 µl of reticulocyte lysate, 0.5 µl Methionine, 2 µl of the reaction buffer. 3 µl of [ 35 S] Methionine (1 mCi/mmole) were added. Reaction was started by adding 1 µl T7 RNA Polymerase and allowed to proceed for 90 min at 30°C. Subsequently, complete samples were filtered through a micro Bio-Spin 6 chromatography column (BioRad). The flow through was analyzed by SDS PAGE and used in protein-lipid overlay assays.

Protein quantification -10 µl of whole cell lysate were diluted in 1 ml of C 2 H 5 OH and incubated on dry ice for 15 min. After centrifugation (15 min at maximum speed in a benchtop centrifuge) the supernatant was discarded and 10 µl of 0.1 N NaOH and 200 µl of micro BCA (Pierce) working solution were added. Following vigorous vortexing and incubation at 37°C for 45 min, 100 µl were transferred to a 96-well plate and read on a Molecular Device SpectraMax 340 PC 384 plate reader set at 562 nm. BSA was used as the standard. Normalisation for CerK protein content, as used for Fig. 5A and Table 1, was obtained by measuring GFP fluorescence of the lysates with a Molecular Devices SpectraMax Gemini XS (Ex/em:395/507 nm; cut-off:495 nm).

Ceramide kinase assays -For in vitro kinase assays, cells were scraped into lysis buffer (10 mM MOPS pH7.2; 2 mM EGTA, 150 mM KCl, 2 % Triton X-100, 1 mM DTT, and protease inhibitors). The suspension was homogenized by 20 strokes in a Potter-Elvejhem homogenizer and used immediately. Kinase activity assays were performed in total volumes of 100 µl composed of: 880 µM C8-Ceramide; 1 mM Cardiolipin; 1.5 % β-Octylglucoside; 0.2 mM diethylenetriamine-pentaacetic acid; 20 mM MOPS pH 7.2; 50 mM NaCl; 10 mM DTT; 2 mM EGTA; 3 mM CaCl 2 and 500 µM [γ-32 P]ATP (40-100 mCi/mmol). Assays were started by the addition of 20 µl of protein sample and vortexing, and allowed to proceed for 15 min at 30°C.

Reactions were stopped by adding 1 ml of freshly prepared CHCl 3 /CH 3 OH (1:1) and 420 µl of 1 M KCl in 20 mM MOPS pH 7.2. After vortexing 400 µl of organic phase were transferred into a new tube and 300 µl of 1 M KCl in 20 mM Mops pH 7.2 were added. Samples were vortexed again and spun down. 200 µl of the final organic phase were collected and counted directly.

For cell-based ceramide kinase assays, 48 hr after transfection, 10 6 CerK transfected COS-1 cells, in a 10 cm diameter dish, were incubated at 37°C for 1 hr with 10 ml fresh DMEM (without serum) and 250 µl of [ 32 P] orthophosphate (10 mCi/ml). Cells were then scraped and spun down, the supernatants discarded, and the pellets washed with 500 µl HBSS supplemented with 10 mM EDTA. After spinning again and discarding supernatants, pellets were resuspended in 0.3 ml of HBSS/EDTA. 1 ml of CHCl 3 /CH 3 OH/HCl (100:100:1) was added and, following vigorous vortexing and subsequent centrifugation, the organic phase was collected. Saponification was performed by the addition of 1 sample-volume of CHCl 3 /CH 3 OH (2:1), 0.25 sample-volume of H 2 0 and a 0.25 sample-volume of 1 M NaOH in CH 3 OH. Samples were vortexed and incubated for 1 hr at 37°C. Finally, the samples were spun down and the organic phase recovered and dried for 20 min at room temperature in a Thermo Electron Corporation SPD Speed Vac ® . Dried lipid films were dissolved in CHCl 3 and analyzed on HPTLC silica plate (MERCK) using CHCl 3 /CH 3 OH /CH 3 COOH (65:15:5) as mobile phase. Plates were dried ,directly exposed to Kodak X-Omat Blue XB-1 films, and processed for autoradiography using an Agfa Curix 60 developer.

Protein-lipid overlays -Protein lipid overlay assays were performed according to published procedures [START_REF] Dowler | Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities[END_REF] using PIP strips and PIP arrays (Echelon Biosciences Inc.). Briefly, membranes were blocked for 1 hr at room temperature in blocking buffer (Tris Buffer Saline containing 0.1 % Tween-20 (TBST), with 2 mg/ml fatty-acid-free BSA). Afterwards, membranes were incubated at 4°C overnight, with proteins of interest in hybridization buffer (blocking buffer supplemented with protease inhibitor cocktail). Membranes were then washed 10 times with TBST and subsequently incubated for 1 hr at room temperature, in blocking buffer, with primary antibody (1:2000 dilution for the FLAG M2 antibody (Sigma) and 1:5000 dilution for the anti-GST HRP coupled antibody (Upstate)). After10 washing steps in TBST, hybridization with the secondary antibody, when required (i.e. after FLAG M2), was performed in blocking buffer with a 1:5000 dilution of anti-mouse Ig antibody linked to horseradish peroxidase (#NA9310V Amersham Biosciences). Finally, membranes were washed as before and processed with LumiGLO (Cell Signalling) for detection.

Western Blot analysis -All fractions were diluted in 3:1 in 4X NuPAGE LDS sample buffer supplemented with 200 mM DTT, and incubated at 75°C for 10 min before snap freezing and storage at -80°C. PAGE analysis was carried out with NuPAGE Bis-Tris 4-12% run in MOPS buffer at 115 V for 2 hr. Gels were transferred to Hybond -ECL™ nitrocellulose membranes (Amersham Biosciences) in a BioRad Trans-Blot SD Semi-Dry Transfer cell (25 V and 100 mA per membrane, for 2 hr) and probed with an anti-GFP antibody (Abcam ab290 rabbit polyclonal) followed by an anti-rabbit Ig antibody linked to horseradish peroxidase (# NA9340V, Amersham Biosciences), or with an anti-FLAG antibody (Sigma, M2) followed by an anti-mouse Ig antibody linked to horseradish peroxidase (# NA9310V, Amersham Biosciences). Detection was carried out with ECL™ (Amersham Biosciences) or LumigGLO (Cell Signalling) using either Hyperfilms ECL™ (Amersham Biosciences) or a Fujifilm Intelligent Dark Box LAS 3000 imager. Band intensities were measured using ImageJ 1.33u (Wayne Rasband NIH http://rsb.info.nih.gov/ij/Java 1.3.1_03).

Tryptic proteolysis -Trypsin was dissolved in 1 mM HCl to 10 µg/ml. COS-1 cell lysates were mixed 3:1 with trypsin dilutions, to achieve final trypsin concentrations of 0.001; 0.01 and 1 µg/ml. Samples were incubated for 30 min at 30°C, and then processed for Western Blotting as described above.

RESULTS

CerK subcellular localization and extractability

Using a GFP-tagged protein, we previously reported that CerK mostly localizes within perinuclear regions and cytoplasmic vesicles upon expression in COS-1 cells as well as HUVEC [START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. Since then we extended this observation to a number of other cell types such as A549, SW1353, CHO and HEK293 (unpublished data). Such a localization has also been reported for endogeneous CerK [START_REF] Lamour | Ceramide-1-phosphate: the "missing" link in eicosanoid biosynthesis and inflammation[END_REF]. In Figure 1A, we used Bodipy-labeled ceramide in order to label the Golgi complex and this showed that the perinuclear CerK molecules indeed co-localize with this organelle. Moreover, many vesicles where CerK was detected also displayed Bodipy fluorescence, an indication that they may originate from the Golgi. Some vesicles instead did not, and thus might be associated with distinct subcellular compartments which remain to be determined. Treatment with nocodazole -a substance known to disrupt microtubules -led to redistribution of CerK within the cell (Fig. 1B). Furthermore, the directed motility of CerKloaded vesicles was lost in cells treated with nocodazole; vesicles showed instead random displacement (see Supplementary Information, Movies 1,2). Nocodazole also hindered the plasma membrane accumulation of CerK that takes place upon hypotonic challenge (Fig. 1B, right). In contrast, treatment with cytochalasin D, which blocks actin polymerization resulting in microfilament breakdown, did not induce relocalization of CerK despite clear morphological changes (not shown). Also, CerK displaying vesicles were still capable of directed movement following this treatment (see Supplementary Information, Movie 3). Therefore, microtubules rather than actin filaments, are required for CerK localization and trafficking.

Before CerK was cloned, it was reported that solubilization of a ceramide kinase activity from brain membranes required prolonged treatment with a combination of detergent, salt and chelator [START_REF] Bajjalieh | Ceramide kinase[END_REF]. Since then, work with recombinant wild-type CerK has shown that CerK mostly associates with the particulate fraction [START_REF] Sugiura | Ceramide kinase, a novel lipid kinase[END_REF][START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. Furthermore, the complex Ca 2+ /calmodulin was recently reported to be important for regulation of CerK activity [START_REF] Mitsutake | Calmodulin is involved in the Ca 2+ -dependent activation of ceramide kinase as a calcium sensor[END_REF]. Therefore, we tested the effects of a neutral detergent, Triton X-100 (or X-114), a monovalent salt, KCl, and a divalent ion chelator such as EGTA, either alone or in combination, on the extractability of CerK. As shown in Fig. 1C, only upon combination of these reagents, could CerK be efficiently released into the supernatant.

Constitutive Golgi/vesicle targeting, requirement for microtubules as well as strong association with the particulate fraction, prompted us to ask for the responsible determinants within the CerK protein.

CerK PH domain: Threading, homology modeling and ESP calculations

The 100-120 amino acid long Pleckstrin Homology (PH) domain belongs to one of the most abundant domain classes in the human genome [START_REF] Mayer | A putative modular domain present in diverse signaling proteins[END_REF][START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF]. Today more than 60 structures from PH domains of various proteins and from different species are known, which are all characterized through a structurally conserved superfold: a seven stranded β-sandwich, capped with a C-terminal α-helix [START_REF] Blomberg | The PH superfold: a structural scaffold for multiple functions[END_REF]. The PH domain of CerK was originally annotated based on a not significant database search hit (E-value 5.4) to the SMART PH domain HMM SM00233 [START_REF] Letunic | SMART 4.0: towards genomic data integration[END_REF]. In order to select a suitable template for homology modeling we performed a fold recognition (threading) analysis with the putative CerK PH domain against the protein structure database (PDB). Upon visual inspection of the sequence-structure alignments of the highest ranking models, we selected the PH domain structure of "Tandem PH domain containing Protein-2" (TAPP2, 1V5P.A) to serve as a template for a low resolution model of the CerK PH domain. This template is the A chain and the best representative conformer of the 1V5P NMR ensemble of 20 structures [42].

The modeled PH domain of CerK displays the typical PH domain superfold (Fig. 2A).

Opposite to the helix are three loops interconnecting β1-β2, β5-β6 and β6-β7. Compared to other PH domains these loops are unusual: β5-β6 and β6-β7 loops are extended and β6-β7 features two additional β-strands, β6' and β6''. Instead, β1-β2 is very short.

Determination of electrostatic potential (ESP) is a valuable tool to predict properties of

PH domains such as their ability to bind membranes [START_REF] Cozier | Membrane targeting by pleckstrin homology domains[END_REF]. As shown in Fig. 2B, CerK PH domain belongs to the subfamily of PH domains with strong molecular polarization. Notably, the extended hairpin-like β6-β7 loop largely contributes to the positive isosurface.

We examined in detail the dependence of the function of CerK on the positively charged amino acid residues of its PH domain (Fig. 3). Either alone or in combination we substituted Arg and Lys residues for Ala or Val (Table 1) and studied the intracellular localization, activity, and biochemical properties of the resulting mutant proteins.

Golgi localization and membrane association are lost in CerK PH domain β6-β7 loop mutants

As shown in Fig. 4A, removal of the PH domain prevents CerK from associating with the Golgi complex. We made use of this clear readout to characterize CerK PH domain mutant proteins. Compared to WT CerK, localization was almost unchanged upon mutagenesis of positively charged residues of either the β1-β2 loop (17/20: K17A and R20A), the β3-β4 loop (29/33/36: R29,33,36A) or the β5-β6 loop (68/74/80: K68,74,80A) of the PH domain. In contrast, mutagenesis of the β6-β7 loop impaired the ability of CerK to localize to the Golgi complex, either partially (90/91: K90A,R91A and 96/98: R96A,K98V) or totally when these two double mutations were combined (90/91/96/98). The latter mutant localized in the cell as would ∆PH-CerK. In comparison, a catalytically defective CerK mutant (Kin -) mutagenized in the ATP binding site (G198D), was correctly localized. These results identify the β6-β7 loop as the key provider of positively charged amino acids to allow for localization of the wild-type protein. A minor role for the β1-β2 as well as β3-β4 loop becomes visible with mutants displaying a partially compromised β6-β7 loop (Table 1). Instead, positive charges within the long β5-β6 loop do not appear to be involved, since their substitution does not aggravate the phenotype of such partially compromised mutants (Table 1).

Differential fractionation was performed to compare WT and mutant CerK proteins (Fig. 4B). Only minute amounts of WT protein could be solubilized in the absence of detergent, whether at low ionic strength (cytosolic fraction) or at high ionic strength (perimembrane fraction). A significant amount of WT CerK was released with the addition of Triton-X-100 to the extraction medium (Triton-extractable fraction; compare also lanes 3 and 4 in Fig. 1C), whereas most of the WT CerK protein was still present in the final pellet (Triton-insoluble fraction). The CerK Kin -mutant protein, which possesses a functional PH domain and localizes as the wild-type enzyme when fused to GFP (Fig. 4A), fractionated very similarly to WT CerK.

In particular, it also displayed membrane extractable material (Triton-extractable fraction). In contrast, neither the CerK ∆PH nor the CerK β6-β7 loop (90/91/96/98) mutant proteins, were recovered from the membrane extractable fraction. This demonstrates that the PH domain is required for CerK to associate with the membrane, and indicates a critical role played by the β6-β7 loop in this process. In the absence of a functional PH domain, the amount of cytosolic protein The half life of CerK enzyme activity was reduced two-fold when comparing the 90/91 mutant to the wild-type protein (Fig. 6B, left). Indeed, 20 min at 30°C decreased the activity of the wild-type protein by 50%, whereas a similar reduction occurred in less than 10 min for the mutant. Accordingly, the inclusion of glycerol significantly slowed the inactivation process, allowing the mutant to approach wild-type activity levels (Fig. 6B, right). Therefore, positively charged residues in the β6-β7 loop of the PH domain allow for conformational stabilization of CerK. This was also exemplified when looking at the trypsin sensitivity of the 90/91/96/98 mutant compared to the wild-type protein. As shown in Fig. 6C trypsin concentrations as low as 0.001 µg/ml result in almost complete disappearance of full length 90/91/96/98 mutant protein whereas wild-type CerK remains undegraded when incubated with 1 µg/ml trypsin for the same period of time.

As seen in Fig. 6D (left) mutation of amino acids 90 and 91 induces a small downwards mobility shift after SDS-PAGE, not seen with any other mutations including those at residues 96 and 98. However, mutations at residues 96 and 98 synergize with those at residues 90 and 91, giving rise to a large downward shift. This synergy was not observed when combining mutations at residues 90 and 91 with mutations on other loops (not shown). Therefore, a fast running behavior typifies mutants with a compromised β6-β7 loop. In vitro translated β6-β7 loop mutants showed a smeary pattern, displaying fast running protein material as well as nearly wild-type-like running material in variable amounts (Fig. 6D,right). This indicates that the mobility shifts observed with β6-β7 loop mutants are not a mere consequence of the net loss of positive charges due to mutagenesis. Furthermore, the increased mobility could not be accounted for by proteolysis, as an N-terminally GFP tagged and C-terminally FLAG tagged 90/91/96/98 mutant protein could be detected by antibodies raised against the two fusion tags (not shown). We also examined if glycosylation could have been counteracted in the β6-β7 mutant proteins, thus leading to the observed fast running species. However, we did not detect any glycosylation in either wild-type or 90/91/96/98 CerK mutant proteins (not shown). Therefore, these experiments rather show that the β6-β7 loop displays important structural features. Mutagenesis of this loop has resulted in changes that are still visible after denaturation, i.e. the mutant protein shows enhanced globularity and thus, reduced apparent molecular size. As shown in Fig. 6D, left, the structural impact of such mutations also gave rise to protein multimers.

DISCUSSION

We made a detailed mutagenesis study of the PH domain of CerK, to identify positively charged residues important for membrane association and Golgi targeting (Fig. 1). Modeling the PH domain of CerK and ESP calculations (Fig. 2 and3) indicate that charged amino acid residues are displayed on four loops β1-β2, β3-β4, β5-β6, and β6-β7, on one face of the PH domain. According to the obtained model, the β6-β7 loop would make critical contribution to the positive ESP and this was indeed evidenced by the mutagenesis study reported herein.

The β6-β7 loop in the PH domain of CerK has conformational impact, effecting both localization (Fig. 4) and activity (Fig. 5 and6). There appears to be a direct correlation between the ability of CerK to bind membrane and to be active since all mutants that are compromised in membrane binding ability also show reduced activity. The converse however does not hold true since an inactive ATP binding site mutant (Kin -), localizes like the WT enzyme. Therefore, catalytic ability is not a prerequisite for membrane binding. We showed that in the absence of a functional PH domain, recovery from a Triton extractable membrane compartment (Fig. 4B) and ability to interact with PPIn (Fig. 4C) are lost. Of note, these mutant proteins unable to bind to the Golgi complex and rather showing diffuse cytoplasmic localization are, for a large part, not cytosolic but rather associate with a Triton X-100 resistant compartment (Fig. 4B). Neither a CAAX sequence added at the C-terminus of ∆PH-CerK nor the fusion of ∆PH-CerK with the Nterminal 20 residus of CerK itself (containing the myristoylation signal) were able to target the resulting ∆PH-CerK to the plasma membrane (data not shown), further evidencing that ablation of the PH domain does not render the protein merely cytosolic. We checked if the accumulation of the ∆PH-CerK protein into detergent resistant compartment might have resulted from misfolding and sequestration into aggresomes. These are nocodazole-dispersible Triton X-100 insoluble specialized structures formed by the condensation of mini-aggregates [START_REF] Garcia-Mata | Hassles with taking out the garbage: aggravating aggresomes[END_REF]. Treatment of cells overexpressing ∆PH-CerK with nocodazole did not have an effect (not shown), therefore we believe that Triton insolubility of CerK proteins is functionally relevant. It is actually possible that other parts of the CerK protein contribute to its localization and this will deserve further studies.

Partial mutagenesis of the β6-β7 loop allowed for studying how it impacts enzyme activity. As shown in Fig. 6A, neither affinity towards ceramide, nor that towards ATP were modified in the 90/91 mutant protein. Rather, β6-β7 loop mutants were found to be destabilized proteins, with reduced catalytic stability (Fig. 6B) and enhanced susceptibility to proteolysis (Fig. 6C). Increased running behavior and multimer formation (Fig. 6D) were also hallmarks of CerK proteins with mutagenized β6-β7 loops, indicative of structural changes, maybe through increased hydrophobic surfaces. Overall, mutants with a compromised β6-β7 loop displayed an unstable conformation which was subject to enhanced deactivation, proteolysis, and aggregation.

Whether PPIn participate in wild-type CerK localization, trafficking and activity will require further investigation. Although early studies had spotted binding to PPIn as characteristic of PH domains, more recent ones have indicated that selective PPIn binding, instead, is not a frequent theme [START_REF] Cozier | Membrane targeting by pleckstrin homology domains[END_REF][START_REF] Lemmon | Pleckstrin homology domains: not just for phosphoinositides[END_REF]. In fact, only in very few cases is PPIn binding by PH domains of high affinity and specificity. This class of PH domains can function independently as signal-regulated membrane-targeting modules. CerK, for a large part, appears to be constitutively targeted to the Golgi complex. Furthermore we previously showed that it can interact with liposomes devoid of PPIn [START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. However, plasma membrane localization also takes place and can be triggered by treatments such as hypotonic shock (Fig. 1B). Therefore, although clearly beyond the scope of the present work, it will be important to study if any of the PPIn identified here using lipid-CerK overlays (Fig. 4C) turns out to be important for CerK biology.

The β1-β2 loop in the CerK PH domain is strikingly short (Fig. 2 and3). Our study actually underscored that its mutagenesis (17/20) has little effect on localization and activity as long as the β6-β7 region is conserved (Fig. 2,4 and Table 1). Nevertheless, a minor role of the β1-β2 region became evident when the β6-β7 region itself is compromised (Table 1). A similar finding was found for the β3-β4 loop (compare mutant 29/33/36/90/91 to mutant 90/91 in table 1). Therefore, all variable loops previously described to impact PH domain behavior, namely VL1 (β1-β2), VL2 (β3-β4) and VL3 (β6-β7) are also shown the be important to CerK. However, whereas the VL1 loop is most critical to other PH domains, our findings reveal that in CerK, VL3 is playing a major role. According to the model presented in Fig. 2, the β6-β7 loop of CerK PH domain ranks among the longest β6-β7 loops when compared to PH domains of known structure.

Of note, this loop is highly charged and would display two additional strands, β6' and β6'', linked by a stretch of intercalated positively charged and hydrophobic residues: KRARRHRWKW, conserved in CerK from all species (Fig. 3). The presence of tryptophans in this region is remarkable. Hydrophobic residues in this motif may serve as an anchor towards the core of the protein to help position the charged residues. Alternatively, they may be used as buffers between charged residues and be critical to penetration of the protein into the lipid bilayer. It will be interesting to study if electrostatic interactions as well as tryptophan insertion contributed by the β6-β7 loop of its PH domain, might be required for CerK membrane binding and interfacial activation, a mechanism that has been described for phospholipases A2 [START_REF] Gelb | Interfacial binding of secreted phospholipases A(2)[END_REF]. This shall also be clarified when the crystal structure of CerK is available.

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060316

CERK construct Localization Activity -/+ SD WT

••• 100 - ∆PH ○○○ <1 - ∆2-7 ••• 60 18 ∆2-13 ○○○ <1 - K17A ••• - - R20A ••• - - K17A, R20A ••○ 77 8 R29A,R33A ••• - - K33A ••• - - R36A ••• - - R29,33,36A ••• 117 13 K74A ••• - - K68V,K74A ••• - - K77A ••• - - K80V ••• - - K74,77A ••• - - K77,80A ••• 102 35 K 68, 74, K80V ••• 114 24 K90V ••• 92 16 R91A ••○ 34 5 K90V,R91A •○○ 19 4 R93,94A ••• - - R96A ••• 103 26 K98V ••• 120 14 R96A, K98V •○○ 61 9 K90V, R91A, R96A, K98V ○○○ <1 - K17A,R20A,K90V, R91A ○○○ 6 3 K17A,R20A,K90V, R91A; R96A, K98V ○○○ <1 - R 29, 33, 36 A, K90V, R91A ○○○ 2 1 K77, 80A, K90V, R91A •○○ 18 8 ∆118-124 ○○○ <1 - β3-β4 + β6-β7 β5-β6 + β6-β7 PH domain region β2-β3 β5-β6 β1-β2 β3-β4 β6-β7 β1-β2 + β6-β7
Table 1: Summary of CerK mutant proteins and associated phenotypes

The CerK mutants are categorized according to the mutagenized region. Localization is depicted using a 3 black-dotted scale: (•••) wild-type, (••○) partially compromised, (•○○) severely compromised, (○○○) ∆PH CerK-type localization. Activity is expressed as a percentage of that of WT-CerK, after normalization of CerK protein amounts (cf Supplementary Fig. 1). Data represent the mean and SD of at least two experiments, performed in triplicates. Deletion of the first 7 amino acid residues of CerK (∆2-7 CerK) has no effect, consistent with the PH domain starting at residue number 8 (Fig. 3) [START_REF] Carre | Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain[END_REF]. Further deletion (∆2-13 CerK) removes most of the first β-strand and completely abrogates wild type localization and activity. An already described L10A mutation [START_REF] Kim | The leucine 10 residue in the pleckstrin homology domain of ceramide kinase is crucial for its catalytic activity[END_REF] was partially effective in our assays. The results obtained with mutagenesis of the loops interconnecting the β-strands, either alone or combined, are summarized. Finally, deletion of the C-terminal α-helix, which abrogates PH domain function, is also indicated. The protein structure was colour-coded from red (N-terminus) to blue (C-terminus).

LEGEND TO FIGURES

FIGURE 3: Structure-based multiple sequence alignment of CerK PH domains

CerK PH domain sequences of multiple species are aligned according to a multiple structure alignment of known PH domains. Highly conserved residues are white on black background, conserved negative residues (DE, conserved marker "-"), positive (HKR,"+"), tiny (AGS,"t"), polar (CDEHKNQRST,"p") are bold on white background, conserved aliphatic (ILV,"I"), hydrophobic (ACFGHILMTVWY,"h"), aromatic (FHWY,"a") and big (EFHIKLMQRWY,"b") residues are set on light grey background (italic font for big residues), conserved charged (DEHKR,"c") residues on dark grey background, and conserved small (DEHKR,"s") residues are shown in grey font [START_REF] Goodstadt | CHROMA: consensus-based colouring of multiple alignments for publication[END_REF]. PPIn arrays, were assayed as described in the experimental section, followed by autoradiography or anti-GST western blotting, respectively; Right, control GST protein domains (see experimental section for full description), probed with anti-GST western blotting [START_REF] Dowler | Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities[END_REF]. Lower panel, phospholipid strip overlay analysis using C-ter FLAG tagged immunoprecipitated proteins: full length CerK, or its PH domain only (CerK-PH), or CerK lacking its PH domain (∆PH-CerK), or the K90V,R91A CerK mutant protein, followed by anti-FLAG western blotting. On the right, the PH domain of PLCδ1 was used as a control. Expression of mutant proteins with a heavily compromised PH domain, reached on average about 50 % of WT CerK levels (see Supplementary Fig. 1). (B) In-cell kinase assays were performed for N-terminally GFP tagged WT CerK and key mutants, using [ 32 P] labeling followed by lipid extraction (densitometric measurements as % relative to WT-CerK are indicated underneath).

FIGURE 6: Biochemical characterization of CerK PH domain mutant proteins

(A) Substrate kinetic studies were performed using C8-ceramide and ATP as substrates and a

CerK construct harboring a 6xHis-FLAG tag at the C-terminus. Wild-type and the 90/91 CerK mutant were overexpressed in COS-1 cells and lysates directly used for the in vitro kinase assays.

For each kinetic a Lineweaver-Burk plot is shown, which was used for evaluation of K m values. This is one of two similar experiments, displayed as means +/-SD for triplicate evaluations. Lysates were incubated with different amounts of trypsin (0; 0.001; 0.01; 1 µg/ml). After a 30min time incubation, samples were processed for PAGE analysis. Western blotting was performed using an anti-FLAG antibody. This is one of two experiments with similar results. (D)

Left, N-terminally GFP tagged CerK and mutant alleles were overexpressed in COS-1 cells. SDS-PAGE was performed on lysates followed by Western Blot analysis using an antibody against the GFP tag. The presence of protein multimers is indicated by arrow heads. Right, in vitro translated

-MGATGAAEPLQSVLWVK---------Q-------QRCAVSLEPAR---ALLRWWRSPG mouse --MGAMGAAEPLHSVLWVK---------R-------RRCAVSLEPAR---ALLRWWRSPE rat --MGAMGAAEPLQSVLWVK---------Q-------QRCAVSLEPAR---ALLRWWRSPE chicken MEQLPGGRRLRLASSLGLG---------Q-------RCWEVTLDDER---GLLA-WRDPR frog MESAHSTSLAVLCSVLSVK---------R-------QSCEVTLDPGR---SLLS-WRELR 1btk.B.- ---------VILESIFLKRSQQKKK (

FIGURE 1 :

 1 FIGURE 1: Subcellular localization and solubilization of wild-type CerK (A) COS-1 cells overexpressing GFP tagged WT-CerK were incubated for 30 min with 5 µM BODIPY ® -TR-ceramide. After medium exchange, cells were analyzed by fluorescence microscopy. (B) COS-1 cells overexpressing GFP tagged WT-CerK were treated or not with 33 µM nocodazole for 3 hours. Subsequently cells were osmotically challenged by diluting the medium 1:4 in water and incubated for another 30 min before fluorescence microscopy. White arrows indicate plasma membrane localization. (C) COS-1 cells overexpressing C-terminally FLAG-tagged CerK were fractionated as described in the experimental section, in the absence or presence of 2 % Triton X-114, 2 mM EGTA or 500 mM KCl, as indicated. At the end of the harvesting period, cells were high speed centrifuged and the resulting supernatants (S) and pellets (P) analyzed with SDS-PAGE and anti-FLAG Western Blotting.

FIGURE 2 :

 2 FIGURE 2: Model and ESP calculations for human CerK PH domain (A) Model of CerK PH domain based on TAPP2 (1V5P.A). (B) ESP calculations. The representation is given in colour-coded iso-surfaces at a counter level of ± 144 mV. The red surface encompasses all coordinates in the vicinity of the protein that exhibit a negatively signed ESP of -144 mV. The blue surface is similar but represents a layer of positive ESPs of +144 mV.

  "*" denote mutagenized residues of the present study. The secondary structure elements of the final model are shown in the last row (E = strand, H = helix), following Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060316the default Chroma consensus string (80 %). The unique and conserved β6-β7 loop of CerK PH domains from various species is boxed.

FIGURE 4 :

 4 FIGURE 4: Subcellular localization, fractionation, and PPIn binding of CerK PH domainmutant proteins (A) COS-1 cells were transiently transfected with plasmids encoding CerK wild-type and mutant alleles N-terminally fused to GFP. 24 hr after transfection, cells were analysed using fluorescent microscopy. Pictures shown are representative of the majority of the cellular population, observed in several experiments. Golgi/cytoplasm fluorescence ratios were determined as described in the experimental section, and are given as the mean of measurements obtained with at least 8 cells. (B) Cell fractionation of wild type CerK compared to a CerK allele lacking the PH domain, to the 90/91/96/98 mutant of this study as well as to the kinase-dead CerK G198D mutant (Kin -). All constructs were 6xHis-FLAG tagged at the C-terminus. Cell fractionation was performed as described in the Methods section and obtained fractions were resolved by SDS-PAGE followed by Western Blot using an anti-FLAG antibody. (C) Lipid protein overlays. Upper panel, Left, in vitro translated and [ 35 S] labeled CerK, or purified GST-CerK, binding to

FIGURE 5 :

 5 FIGURE 5: Activity of CerK PH domain mutant proteins (A) Different mutant alleles of CerK were expressed in COS cells and assayed in vitro as crude cell lysates, in comparison to wild-type CerK. Data represent a mean of at least 3 experiments (-SD), performed in triplicates. *** indicates statistical significance with p<0.001 (t test).

(

  •) CerK WT (○) CerK 90/91 (B) Left, Whole cell lysates of COS-1 cells overexpressing WT CerK (-) or the CerK 90/91 mutant (---), C-terminally FLAG-6xHis tagged proteins, were preincubated at 30°C for the indicated times, before assaying for the remaining activity. Right, The presence of 20 % glycerol (grey bars) prevents the inactivation of the CerK 90/91 mutant protein. This is one of two experiments with similar results. (C) Wild type and mutant CerK, both Cterminally 6xHis-FLAG tagged were overexpressed in COS-1 cells and harvested in lysis buffer.

[ 35 S

 35 Fig.2A,B

Fig. 5 Biochemical

 5 Fig. 4A

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060316

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060316 Copyright 2006 Biochemical Society

ACKNOWLEDGMENTS

We are very grateful to Pr. Mark Lemmon (University of Pennsylvania) who kindly provided us with the plasmids used for control overlay experiments. We thank Dr. Michael Forstner (Novartis Institutes for BioMedical Research, Basle) for providing purified GST-CerK, Lucia Schoderböck for help in the preparation of CerK mutants, and Susan DiClemente for editing the manuscript.

directed mutagenesis kit (Stratagene). SDS-PAGE was done under reducing conditions on NuPAGE ® polyacrylamide gels (Invitrogen). OneShot ® TOP10 Biotech Services. Purified insect cell expressed GST-CerK was obtained from M. Forstner (NIBR, Basle).

was somewhat increased. Furthermore and surprisingly, PH domain mutant proteins were still largely present at the particulate fraction.

In order to look for possible regulation of CerK localization by polyphosphoinositides (PPIn) binding to its PH domain, we used PPIn-spotted nitrocellulose membranes and recombinant CerK proteins. Whether expressed in vitro as a untagged protein or produced as a Nterminal GST fusion protein and purified, CerK was found to bind various PPIn: PtdIns(3,5)P 2 , PtdIns(3)P, PtdIns(5)P and Ptd/Ins(4)P (Fig. 4C, upper panel, left). To ensure the quality of the overlay procedure, several control proteins, of known and varied PPIn binding specificities, were tested in parallel (Fig. 4C, upper panel, right) [START_REF] Klein | The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding[END_REF][START_REF] Sankaran | High-affinity binding of a fyve domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not fyve domain oligomerization[END_REF][START_REF] Kavran | Specificity and promiscuity in pleckstrin homology (PH) domain binding to phosphoinositides[END_REF]. The isolated PH domain of CerK displayed a PPIn binding selectivity identical to the full length protein (Fig. 4C, lower panel). In addition, both could bind phosphatidic acid (PA). In the absence of PH domain, a dramatic loss of binding to all PPIn (and PA) was observed; accordingly, partial mutagenesis of the β6-β7 loop (90/91) only had partial impact (Fig. 4C, lower panel). These experiments indicate that an intact β6-β7 loop is necessary for the PH domain to mediate CerK binding to PPIn.

CerK PH domain β6-β7 loop mutants are functionally compromised

CerK activity was measured in vitro (Fig. 5A). All mutants displaying wild-type localization (as seen in Fig. 4) also displayed wild-type catalytic activity (29/33/36, 68/74/80); those with partial loss of localization had lower activity (e.g. 90/91, 90/98); finally the mutants with the most aberrant localization showed the weakest activity (90/91/96/98). Therefore it seems that loss of localization parallels loss of activity. Cell-based CerK assays were performed to test compromised mutants more directly. Consistent with the in vitro assay, the 90/91 mutant showed partial activity whereas the 90/91/96/98 was totally devoid of activity (Fig. 5B).

In order to characterize how the PH domain of CerK impacts activity, we studied the partially compromised 90/91 mutant with regard to substrate affinities. As can be seen in Fig. 6A, affinities of this mutant CerK protein towards either ceramide or ATP were similar to those of the wild-type protein (WT-CerK: K m = 205 µM and 52 µM ; 90/91-CerK mutant: K m = 194 µM and 42 µM for ceramide and ATP respectively), in line with published data [START_REF] Sugiura | Ceramide kinase, a novel lipid kinase[END_REF]. Hence, the PH domain of CerK influences enzymatic activity through a mechanism that does not involve substrates recognition.


Jacqueline Dunne

Alexis Caron

Patrick Menu

Abdu I Alayash $

Paul W Buehler $

Michael T Wilson #

Radu Silaghi-Dumitrescu #

Beatrice Faivre

Professor Chris E Cooper
email: ccooper@essex.ac.uk

Ascorbate removes key precursors to oxidative damage by cell free hemoglobin in vitro and in vivo

Keywords: hemoglobin, blood substitute, ascorbate, free radical, oxidative stress, ferryl Biochemical

Hemoglobin initiates free radical chemistry. In particular the interactions of peroxides with the ferric(met) species of hemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell free hemoglobin stabilized in tetrameric form (via crosslinking with bis(3,5dibromosalicyl)fumarate). The transfusate contained 95% oxyhemoglobin, 5% methemoglobin and 25 µM free iron. Electron paramagnetic resonance spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methemoglobin was reduced to oxyhemoglobin by a slower process (t 1/2 = 1 hour). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methemoglobin, ferryl hemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intraerythrocyte ascorbate as electron donor). As well as relating to the toxicity of hemoglobin-based oxygen carriers, these findings have implications for situations where heme proteins exist outside the protective cell environment e.g. hemolytic anemias, subarachnoid hemorraghe, rhabdomyolysis.

(a) INTRODUCTION

Hemoglobins and myoglobins are involved in oxygen transport and storage. As a consequence of this function and because of the redox properties of the iron these proteins also generate reactive oxygen species [1]. Autoxidation of the oxy derivative (Fe 2+ ) leads to non-functional ferric heme (Fe 3+ ) and superoxide ion (O 2

•-), which subsequently dismutates to generate hydrogen peroxide (H 2 O 2 ). These species can ultimately damage the protein and/or the heme group [2]. An essential intermediate in the pathway leading to this damage is the ferryl heme (Fe 4+ ), itself formed through the reaction of the ferric heme with H 2 O 2 [3]. Accompanying the formation of the ferryl heme [4,5] is a protein/porphyrin-based radical cation (P +• ) (see Eq 1).

P-Fe 3+ + H 2 O 2 → P +• Fe 4+ =O 2-+ H 2 O Eq. 1

Ferryl heme and the radical can also be extremely toxic, notwithstanding their transient existence. These oxidative cascades can be damaging because a) peroxide is a powerful oxidant known to produce cellular damage [6] b) both the ferryl heme and protein-based radicals can initiate oxidation of lipids, nucleic and amino acids by abstraction of hydrogen atoms [7,8] (c) heme modification can lead to highly toxic heme to protein crosslinked species and to the loss of heme and the release of the "free" iron [2,[9][10][11].

The potential for hemoglobin-mediated peroxidative damage exists whenever the protein is removed from the protective environment of the erythrocyte. This would occur for example during spontaneous erythrocyte hemolysis or in haemolytic anemias (e.g. sickle cell anemia [12]). We have shown that myoglobin induces kidney damage following crush injury (rhabdomyolysis) by exactly this peroxidative mechanism rather than free iron catalyzed Fenton chemistry as was previously thought [13,14]. In contrast, there has been very little research carried out in animals or humans on hemoglobin oxidative kinetics and possible contributions of these reactions to the overall toxicity of free hemoglobin. However, we have recently shown that hemoglobin can cause similar Copyright 2006 Biochemical Society damage in vivo when it is released from the erythrocyte in subarachnoid haemorrhage [15]. Furthermore, uncontrolled heme-mediated oxidative reactions of cell-free hemoglobin (developed as a blood substitute) have emerged as an important potential pathway of toxicity, either directly or via interactions with cell signalling pathways [16].

The toxicity of ferryl hemoglobin has been demonstrated in an endothelial cell culture model system of ischemia/reperfusion [17] and in cells that lacks their antioxidant mechanisms such as glutathione [18]. Ferryl hemoglobin can cause cell injury, including apoptosis and necrotic cell death. Perfusion of rat intestine with chemically modified hemoglobin has been shown to cause localized oxidative stress leading to leakage of the mesentery to radio labelled albumin [19]. Importantly, the cynaomet derivative of this hemoglobin in which the heme iron is blocked with cyanide and is unavailable to enter redox reaction produced no cellular changes.

In spite of the presence of a number of well-known antioxidant defence mechanisms very little is known about the precise mechanism(s) by which hemoglobin oxidative toxicity is controlled in vivo. In vitro, the properties of ascorbate (vitamin C) as a reductant are well established in a range of biological systems [20]. Reperfusion of isolated ischemic rat hearts with ascorbate decreased myocardial injury [20,21]; other investigations have shown that ascorbate directly suppressed ferrylMb in isolated hearts [22]

Hemoglobin solutions

For transfusion studies 8.2g.dL -1 solution of hemoglobin crosslinked between the alpha subunits with bis(3,5-dibromosalicyl)fumarate in Ringers lactate (Hb-DBBF, Walter

Reed Army Institute of Research, Washington, DC, USA). For the in vitro studies both Hb-DBBF and "control" HbA o were used. The latter was prepared from human volunteer blood by the method of Antonini [25] with the addition of ion exchange chromatography on DEAE Sephadex A50 [26] to remove contaminating catalase. HbA o was stored in the carbon monoxide bound form at 77K to prevent autoxidation. Methemoglobin was prepared from carbonmonoxy hemoglobin as described previously [27].

Preparation of Erythrocytes and Erythrocyte Ghosts

Whole blood was collected in ethylenediaminetetraacetic acid (EDTA) from healthy volunteers under informed consent and with approval of the National Institutes of Health, Institution Review Board (protocol # 03120B). Whole blood in EDTA was washed with pH 7.4 PBS buffer, centrifuged at 3000 rpm followed by removal of non-erythrocyte components. The process was repeated five times to isolate packed erythrocytes. Ghost erythrocytes (unsealed) were prepared according to the methodology described by Steck and Kant [28].

Reduction of MetHb-DBBF by Erythrocytes and Erythroycte ghosts

Aliquots of erythrocytes or ghost erythrocytes were prepared in 1.5 mL conical tubes (n=3 tubes per time point) and diluted appropriately to simulate 0, 20, 30 and 40%

Isovolemic hemodilution in anesthetized rabbits

The animal protocol was approved by the French National Ethics Committee (licence No.

006101), and the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1985). Male New Zealand white rabbits (La Garenne, Villey Saint-Etienne, France, weighing 2.7 ± 0.3 kg)

were acclimatized for 1 week after arrival with free access to food and water. At the end of the experiments, the animals were sacrificed by an excess dose of pentobarbital. Three hours prior to the study the animals were anesthetized with halothane (Blamont, Neuillysur-Seine, France) and chronically implanted with 2 venous and 2 arterial heparin-filled polyethylene catheters. The conscious animals were then subjected to exchange transfusion (20% of total blood volume) with saline (n=7) or Hb-DBBF solution (n=6).

Mean arterial pressure (MAP), heart rate (HR), blood pH, haemoglobin concentration (Hb tot ) and hematocrit (Hct) were monitored before exchange transfusion and for a 3-hour period of isovolemic hemodilution. The duration of the exchange transfusion was approximately 30 minutes; the initial time points when samples were taken posttransfusion (t = 5) refers to the time after the end of this period (and therefore some in vivo changes will have occurred due to DBBF injected at the beginning of the transfusion period).

Electron Paramagnetic Resonance (EPR)

EPR spectral measurements were made on blood samples which were collected in sterile heparinized syringes.

HPLC analysis of peroxide-modified hemoglobin

MetHb-DBBF was reacted with hydrogen peroxide and analyzed for the loss of the heme and the formation of crosslinks between the heme group and the globin molecule (termed Hb-H) by reverse phase HPLC using a method, adapted from that of Osawa and Korzekwa [31] and described in detail in [10].

Statistical analysis

Statistical analysis was performed using Graphpad Prism version 3.0 for Windows (Graphpad Software, San Diego, California, USA). The immediate effect of the transfusion was tested by a paired t-test, pre and post transfusion. Subsequent changes post transfusions with time were tested using a one-factor ANOVA for repeated

(c) RESULTS

The effect of peroxides on hemoglobin can be assayed by optical and magnetic techniques. Figure 1a shows the effect of adding a two molar excess of H 2 O 2 (mole H 2 O 2 per mole heme) on the optical spectrum of ferric(met) HbA o . Peaks appear at 545 and 585 nm, characteristic of oxidation of the ferric (Fe 3+ ) to the ferryl (Fe 4+ =O 2-) redox state [32].

Production of the ferryl species was confirmed by the addition of sulfide, which generates sulfhemoglobin from ferryl, indicated by the peak at 620 nm [22]. The addition of H 2 O 2 to Hb-DBBF generates an identical spectrum to that of ferryl HbA o (Figure 1b). The stoichiometry of ferryl formation is also identical in the two hemoglobins (Figure 1c)

with one molecule of H 2 O 2 converting one molecule of met (Fe 3+ ) to ferryl (Fe 4+ = O 2-).
Therefore although H 2 O 2 is a two electron oxidant, only one electron is used to oxidise the heme. The other electron oxidises the protein, generating a protein radical [27]. This radical can be detected magnetically, by the use of electron paramagnetic resonance spectroscopy (EPR).

Methemoglobin has a high spin ferric heme, with characteristic EPR spectral features at g = 6 and g = 1.99 (Figure 1d). Following the addition of H 2 O 2 the heme is oxidised to ferryl (which is low spin and EPR undetectable). Hence there is a decrease in the g = 6 signal. Accompanying this decrease there is a formation of free radicals on the globin, indicated by the sharp signal at g = 2. This spectrum is, in fact, composed of a complicated mixture of radical species situated on tyrosine and tryptophan residues [27,[33][34][35]]. An expansion of the g = 2 region shows that the radical species formed in HbA o are all also formed in Hb-DBBF (Figure 1e). Hb-DBBF therefore is capable of forming both ferryl iron and free radicals in vitro. The overall kinetic profile of the formation and decay of the Hb radicals is not fundamentally altered compared to HbA 0 [36]. However, at 30s post peroxide addition, the concentrations of both the tyrosine radical (+40%) and To explore the likelihood of these species being formed in vivo, exchange transfusions were performed in a conscious rabbit model (n=6). Comparisons were made of an approximate 20% (blood volume) exchange transfused with Hb-DBBF, compared to controls, reinfused with autologous blood (Table 1). The measured hematocrit fell by 23% in the Hb-DBBF animals, but was unchanged in the controls. The basic physiological results were consistent with previous studies [38,39]. Immediately following transfusion there was a significant drop in the heart rate in the Hb-DBBF animals and a rise in blood pressure. These effects were not seen in the control animals and are consistent with the known hypertensive effects of Hb-DBBF, believed to be due to its direct reactions with nitric oxide [40]. Blood pH did not change significantly in either control or Hb-DBBF-treated animals. Total hemoglobin fell slightly in Hb-DBBFtreated animals (immediately following the transfusion), due to the concentration of hemoglobin in the transfused Hb-DBBF solution (8.2 g/dl) being lower than the average concentration in the blood (14.0 g/dl). All the significant changes in the hematocrit, hemoglobin concentrations, heart rate and blood pressure that occurred with Hb-DBBF occurred during the time of the transfusion. No changes were seen in these parameters over the remaining three hours of the study.

EPR measurements on whole blood can reveal information about a range of paramagnetic species, such as free radicals and transition metals. All the EPR data reported here are illustrated by spectra from individual animals, but identical changes were seen in all animals (Table 1). Figure 2a shows the five compounds readily identifiable by EPR in whole blood. They correspond to iron in methemoglobin, catalase and transferrin, copper in ceruloplasmin and the globin free radical(s). Comparisons with plasma and erythrocyte spectra confirm that the catalase, methemoglobin and free radical signal are only seen in erythrocytes and the transferrin and ceruloplasmin signals are only seen in plasma. We have previously identified the free radical signal as being primarily attributable to the tyrosine radical associated with the addition of H 2 O 2 addition to haemoglobin [41] i.e.

there is direct in vivo evidence that within the erythrocyte the reactions outlined in Figure 1 result in a detectable steady state level of globin-bound free radical even in control animals or healthy human volunteers [42]. Copyright 2006 Biochemical Society A comparison of the spectrum of whole blood before and after the exchange transfusion with Hb-DBBF shows a large increase in the methemoglobin signal at g=6 (Figure 2 b,c).

In the plasma pre-transfusion there is, as expected, no g=6 methemoglobin EPR signal (Figure 2d). Following transfusion a similar increase is seen here, as was observed for the whole blood. Although most (>90%) of the Hb-DBBF (pre-transfusion) is present in the oxyhemoglobin form, 5.1 ± 1.2 % is present as methemoglobin. This corresponds to approximately 220 µM methemoglobin in the transfused solution. Therefore a 20% exchange transfusion would be expected to generate approximately 40-50 µM methemoglobin. These levels are equivalent to those observed in the plasma (Table 1).

Thus the data are consistent with the simple explanation that the methemoglobin in the transfused Hb-DBBF solution appears in the plasma, essentially unchanged, at least initially (Note that after the transfusion is over methemoglobin decreases. The discrepancy between the 40 -50 µM methemoglobin in the injected solution and the 33 µM that we observe at t = 0 can be explained by the reduction of methemoglobin injected early in the 30 minute transfusion process).

The only other significant change (Table 1) in the EPR spectra immediately post transfusion is an increase in the iron loading of plasma transferrin (resulting in an increase in the g=4.3 signal). The data could be consistent with the transfusion resulting in a release of iron from erythrocytes or damage to the Hb-DBBF. However, following the initial increases the g=4.3 transferrin signal was unchanged with time (Table 1), suggesting that the transfusate itself was the source of this iron. The Hb-DBBF solution contains only very small amounts of EPR detectable non-haem iron that could not account for this signal directly (<0.05 µM, results not shown). However, the data are consistent with the presence of EPR undetectable low molecular weight iron loosely bound to the protein solution. Upon transfusion this iron would be rapidly scavenged by apotransferrin, producing the increase in the transferrin EPR signal. We tested this by adding the Hb-DBBF solution to rabbit plasma outside the animal (at the same 20% dilution as if it were being added in vivo). There was a significant increase in the iron or EPR parameters in the control animals, following the re-transfusion of autologous blood (Table 1).

Thus the addition of Hb-DBBF to an animal adds two forms of iron, that can potentially initiate free radical damage. The free iron (5 µM) can be effectively detoxified via binding to transferrin. However, the methemoglobin "load" (50 µM) would be expected, if anything, to increase with time due to autoxidation of the remaining 95% of oxyhemoglobin. However, Table 1 shows that the EPR-detected methemoglobin concentration in plasma decreases with time following the transfusion with a half time of approximately one hour. This decrease must arise from a reductive, degredative or transport process. A degredative process would be expected to cause an increase in iron bound to transferrin with time which was not seen; a transport process (actively removing metHb-DBBF from the plasma) seems unlikely -total Hb-DBBF remains unchanged over the relatively short time course of the study and the transport system would have to specifically target methaemoglobin, which seems structurally implausible. Therefore we view the most likely explanation as a reductive process in the plasma, converting the metHb-DBBF to oxyHb-DBBF. Copyright 2006 Biochemical Society (results not shown). Ascorbate, however, is able to reduce metHb-DBBF to oxy (Figure 3a), but at a rather slow rate. The second order rate constant (at 37°C in PBS, pH 7.4) was calculated to be 0.2 M -1 s -1 . This rate requires unphysiological plasma ascorbate concentrations (in excess of 500 µM) to be able to explain the observed reduction of methemoglobin in vivo seen in Table 1. However, the presence of erythrocytes appeared to increase the efficiency of ascorbate reduction of metHb-DBBF (Figure 3b). The effect required whole cells, and could not be reproduced by erythrocyte membranes (ghosts) alone (Figure 3c). This agrees with previous findings that the role of the erythrocyte in catalysing extracellular methaemoglobin reduction by ascorbate is due to the recycling of dehydroascorbate to ascorbate, rather than a specific erythrocyte membrane enzyme. [45,46]. Erythrocytes alone had a negligible ability to reduce metHb-DBBF, in the absence of exogenous ascorbate (Figure 3d).

Detailed analysis of the plasma EPR spectra before and after transfusion revealed an anomalous finding (Figure 4). The only change in the g=2 region related to the features attributable to the high field components of the methemoglobin EPR spectrum (at g=1.99). When these features are removed (via subtracting an appropriate pure hemoglobin spectrum) the remaining spectrum only contained features attributable to the copper centres in ceruloplasmin. In contrast to the situation inside the erythrocyte, where the globin free radical can clearly be seen (Figure 2), we see no detectable protein free radical bound to Hb-DBBF in the plasma. This is despite higher concentrations of metHb-DBBF in the plasma (compared to the erythrocyte) and, presumably, significant peroxide formation due to autoxidation of Hb-DBBF and/or other oxidative stress mechanisms [47].

The difference cannot be due to an intrinsic difference in Hb-DBBF compared to HbA o .

Both have similar autoxidation rates (Hb-DBBF is slightly higher than HbA o [48]) and both form similar radicals in vitro following peroxide addition. Therefore we tested whether plasma ascorbate could scavenge the H 2 O 2 -induced free radical on Hb-DBBF.

Figure 5 shows that the free radical formed on pure Hb-DBBF by H 2 O 2 becomes undetectable in the presence of ascorbate. Instead a spectrum with identical features to the ascorbyl radical is observed. In low temperature experiments (Figure 5a) this is characterised by a narrow 9G singlet with anomalous power saturation characteristics

[49]; at room temperature (Figure 5b) a doublet is observed [50]. The only detectable EPR radical signal formed following H 2 O 2 addition to metHb-DBBF in plasma has identical features to that of the ascorbyl radical (Figure 5c). Removal of plasma ascorbate via pre-treatment with ascorbate oxidase resulted in the appearance of non-ascorbyl radicals (Figure 5d); the exact species responsible for this symmetrical radical (g = 2.004)

is not clear. However, it is inconsistent with low molecular weight urate or glutathionebased radicals. This confirms that it is the presence of ascorbate in plasma that makes it impossible to observe protein radical EPR spectra in the rabbit plasma following transfusion.

Plasma ascorbate also has the potential to reduce the other oxidising species formed following H 2 O 2 addition to methemoglobin, ferryl iron [51]. Figure 6 demonstrates that the addition of ascorbate to ferryl Hb-DBBF causes a rapid formation of met Hb-DBBF.

This reactivity exhibits saturating behaviour with respect to ascorbate with an observed V max of ~50 s -1 (data not shown). Subsequently metHb-DBBF is converted to oxy Hb-DBBF by the slow process previously described (Figure 3). In the steady state ascorbate keeps the concentration of ferryl iron in globins at levels that are essentially undetectable optically [52,53]; this effect is mediated solely by an effect on ferryl reduction, as ascorbate has no effect on the rate constant for ferryl formation from H 2 O 2 and MetHb-DBBF (results not shown).

Ferryl iron and globin free radicals can react together to induce oxidative modifications of the heme that are detectable in normal human blood and are increased under conditions of oxidative stress [47]. More severe oxidative conditions (requiring high peroxide or lowered pH) result in a covalent heme to protein bond [2,10,48,54]. This species (Hb-H) is indicative of enhanced ferric:peroxide reactivity in vivo [15]. Figure 7 shows that physiological levels of ascorbate decrease the formation of Hb-H in vitro. This is even the case at acid pH, where the reactivity of the ferryl iron is at its greatest in vitro and in vivo. We were unable to detect Hb-H in vivo in the control or Hb-DBBF transfused Copyright 2006 Biochemical Society

(d) DISCUSSION

This paper reports on the effect of infusing a high dose of a heme protein into plasma. In particular we have focused on the heme redox chemistry as we have previously shown that this is responsible for the kidney damage due to massive myoglobin release in rhabdomyolysis [14]. Heme-mediated oxidative side reactions of cell-free Hb have emerged as an important pathway of toxicity, which may have possibly contributed to recent clinical setbacks with use of hemoglobin-based products as blood substitutes [16] The spectrosocopic approach adopted informs both on the hemoglobin itself and, directly and indirectly, on the response of the plasma antioxidant defences to the "heme insult".

We show that in the presence of peroxide, Hb-DBBF undergoes an identical stoichiometric conversion from the ferric to the ferryl redox state as occurs with HbAo.

H 2 O 2 accepts two electrons from ferric hemoglobin (see equation 1). One converts ferric to ferryl, the other results in the formation of a cationic radical. The nature of this radical is complex. Unlike the situation with peroxidases and catalases that are designed to react with H 2 O 2 , and maintain the radical in a controlled environment to catalyze subsequent oxidation reactions [5], the radical formed in the "pseudoperoxidase" activity of myoglobin and hemoglobin is less stable and resides on tyrosine and tryptophan amino acids in the globin polypeptide chain [27,[33][34][35]55]. In this paper we report that the EPR spectrum of Hb-DBBF following H 2 O 2 addition is identical to that seen with HbA o . Both the tyrosyl and the tryptophan peroxyl radicals can be detected, confirming that the crosslinking of alpha subunits of Hb-DBBF (engineered to keep the protein in the circulation) do not affect the nature of the redox chemistry at the heme site, nor the subsequent radical migration.

The EPR spectrum of whole blood allows us to measure the ferric redox state of hemoglobin in vivo (g=6 peak). The erythrocyte (catalase) and plasma (ceruloplasmin)

antioxidant enzymes also have EPR detectable ferric iron and cupric copper respectively, allowing quantitative measures of these enzyme concentrations to be determined.

Apotransferrin is EPR silent, but once iron binds a distinct EPR signal appears, allowing Copyright 2006 Biochemical Society the release of iron into the plasma to be monitored. Finally, and most importantly, the free radical bound to globin (and produced via the spontaneous reaction of peroxides with methemoglobin) can also be detected inside the erythrocytes. A single EPR spectrum can therefore simultaneously and quantitatively inform on all significant paramagnetic species in the blood.

What are the changes in the paramagnetic species in blood following a 20% transfusion of a hemoglobin based blood substitute? The clearest change is the increase in the methemoglobin signal. We show that this is entirely due to met Hb-DBBF in the plasma and, pre-existing in the solution prior to transfusion. This level (5%) is within the normal acceptable range for a blood substitute and results from the balance between the rate of reduction and autoxidation during its manufacture and storage [43]. Nevertheless even this level of methemoglobin has the potential to catalyze oxidative damage. In addition to the change in methemoglobin, EPR demonstrates that there is an increase in iron binding to transferrin consistent with a 25 µM concentration of loosely bound iron in the Hb-DBBF pre-infusion. Following the 20% transfusion, the 5 µM "free iron" is rapidly bound to transferrin where it becomes redox inactive. The level of iron binding to transferrin does not increase during the course of the study, and remains well below full saturation. Iron is not released from Hb-DBBF to any significant extent in the three-hour period post transfusion. Therefore Fenton chemistry (where free ferrous iron reacts with H 2 O 2 to form the strongly pro-oxidative hydroxyl radical, OH°) is highly unlikely to play a direct role in the plasma following heme protein release, either in spontaneous hemolysis, haemolytic diseases or following the infusion of a hemoglobin based blood substitute.

The most significant finding in this study is, paradoxically, one relating to the absence of a free radical EPR signal. Throughout the study the globin radical in the erythrocyte remained at a constant concentration throughout the study in both the control and Hb-DBBF transfused animals. However, no free radical was present in the plasma following Hb-DBBF treatment, despite the fact that we know that Hb-DBBF makes identical (and therefore readily detectable) radicals to HbA 0 . The absence of any spectral features Copyright 2006 Biochemical Society attributable to such a sharp feature in the EPR spectrum implies that the concentration must be held at less than 5 nM. We demonstrated that this was entirely consistent with ability of plasma concentrations of ascorbate to scavenge this radical. Furthermore ascorbate had the ability to directly reduce the ferryl Hb-DBBF, in agreement with previous studies on HbAo and myoglobin [51]. Ascorbate is therefore able to chemically reduce both of the toxic oxidants formed following peroxide addition to erythrocyte free hemoglobin (described in equation 1).

Ascorbate not only has the ability to deal with the oxidative products of equation 1, it can also decrease the concentration of the substrate, methemoglobin [56]. However, unlike the situation with the oxidative products this does not involve simple chemistry, but requires the presence of the erythrocyte. As Hb-DBBF cannot readily enter the erythrocyte, this suggests the possibility of a methemoglobin reductase system acting at the external face of the erythrocyte membrane, ultimately driven by electrons from plasma ascorbate. Trans plasma membrane reductase systems in the erythrocyte have been characterised [57,58]. These redox pathways are able to take electrons from intracellular ascorbate (or NADH) and reduce external ferricyanide [59]. However, methaemoglobin itself cannot be reduced directly by this system. Our studies reveal that erythrocyte ghosts do not enhance metHb-DBBF reduction, whilst earlier work demonstrated that reduction occurs even when a dialysis membrane separates the protein and the erythrocyte solutions [45].

Recent studies have suggested that the physiological electron acceptor for the trans plasma membrane reductase is in fact the ascorbyl radical [60,61]. This appears to be a high capacity system -up to 35 µM external ascorbate can be regenerated in blood every three minutes [46]. The activity of this system would therefore be more than enough to maintain a constant steady state extracellular ascorbate concentration in our in vitro study (Figure 3b). This relatively fast rate of ascorbate regeneration, followed by the rate limiting step of ascorbate oxidation by metHb, also explains why increasing the hematocrit above 20% has no effect in our study; as even this low hematocrit should be Copyright 2006 Biochemical Society plasma we do not see high concentrations of the ascorbyl radical, despite our prediction of significant ascorbate oxidation by metHb-DBBF.

The most likely role for the erythrocyte in defending against blood substitute-induced methemoglobinemia is therefore to maintain the plasma ascorbate concentration constant despite the presence of high concentrations of extracellular methemoglobin. The reduction of metHb by ascorbate is linearly dependent on ascorbate concentration and any significant decrease in plasma ascorbate would significantly decrease this rate.

Extrapolating from in vitro reduction rates to in vivo metHb levels is not trivial e.g.

oxyHb-DBBF will be continually regenerating metHB-DBBF due to autoxidation.

However, the data suggest simple reduction by plasma ascorbate as being a dominant mechanism. This role of ascorbate may go beyond the problems of extracellular methemoglobin in blood substitutes. For example the increase in methemoglobin formation following decreases in ascorbate levels after air blast overpressure injury is also consistent with a role for ascorbate in preventing methemoglobinemia [62]. Copyright 2006 Biochemical Society at higher transfusion levels. However, the products of this comproportionation reaction are two molecules of metHb-DBBF, making the role of ascorbate in reducing methemoglobin even more important.

The studies following ascorbate oxidase addition to rabbit plasma demonstrate that ascorbate is the primary low molecular weight antioxidant when dealing with aqueous radicals in rabbit plasma. We were not able to identify low molecular weight thiyl or urate [63] radicals in this system. However, we caution against a simple translation of these findings to the human system. Urate levels [64] are significantly higher in primates than rodents (reaching up to up to 300 µM); although ascorbate is still the dominant scavenger of aqueous radicals in human plasma [66], at these levels urate is more effective than ascorbate at scavenging ferryl Hb ([65] and results not shown).

The proposed multifunctional role of ascorbate in limiting the redox toxicity of heme proteins (as a radical scavenger and a ferric and ferryl heme iron reductant) would be expected to function irrespective of whether the heme protein was released into the plasma as part of normal physiology/pathophysiology, or following the infusion of a hemoglobin based blood substitute. However, this paper has specific implications for the clinical use of blood substitutes: (i) the co-administration of reducing agents, such as ascorbate, may offer a simple strategy to control or reduce hemoglobin-mediated toxicity;

(ii) the hematocrit should not be dropped too low as, even if oxygen transport is adequate, the decrease in erythrocyte concentration could compromise methemoglobin reduction.

(e) ACKNOWLEDGEMENTS

We acknowledge funding from a Wellcome

  and improved cardiac function in a model of magnesium deficiency [23]. Consistent with these observations, recent experiments demonstrated direct correlation between the ascorbatemediated reduction of ferrylMb and hemoglobin and inhibition of apoptosis in culture of endothelial cells exposed to peroxide [24].The infusion of cell-free hemoglobin as blood substitutes in large quantities provides an opportunity to explore the interplay between hemoglobin oxidative side reactions and plasma antioxidant mechanisms. In this paper we report for the first time the results of studies designed to monitor the oxidative consequences of administering cell-free hemoglobin into an animal model of partial exchange transfusion and reveal a novel multifunctional role of ascorbate in preventing heme toxicity in vivo.

  the tryptophan peroxyl radical (+70%) are significantly increased in the case of Hb-DBBF when compared to HbA o , consistent with previous measures of a slightly enhanced rate of reactivity of peroxide with Hb-DBBF[37].

  concentration bound to transferrin (from 18.5 ± 1.0 µM to 23.5 ± 2.0 µM, n = 6, p < Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341Copyright 2006 Biochemical Society 0.001). The mean difference in ferric transferin observed in this in vitro experiment (5.0 ± 0.5) is consistent with that seen in vivo (5.74 ± 0.93 µM), demonstrating that the additional iron bound to transferrin in vivo can be entirely explained by free iron initially present in the Hb-DBBF transfusate. The 25 µM readily chelatable iron in the Hb-DBBF solutions used here probably represent a subset of the total labile iron, calculated to be about 80 µM in solutions of Hb-DBBF[43].The other EPR signals illustrated in Figure2a-ceruloplasmin, catalase and the erythrocyte globin radical(s) did not significantly change during the transfusion process, nor for the three hours subsequent. No significant changes were seen in any physiological

  81.92 ms, scan rate 23G/s. (b) venous blood pre transfusion. (c) venous blood post transfusion. (d) venous plasma pre transfusion. (e) venous plasma post transfusion.

Figure 3

 3 Figure 3 Reduction of metHb-DBBF to oxyHb-DBBF by ascorbate in vitro and in vivo (a) Addition of ascorbate (1.5 mM) to metHb-DBBF (50 µM) in PBS at 37°C. Lines illustrate spectra pre addition and 30 and 60 minutes post addition. Arrows indicate change in absorbance with time. (b) Reduction of 100 µM MetHb-DBBF by 100 µM ascorbate in the presence of varying concentration of erythrocytes. Control (100 µM MetHb-DBBF alone) (■); + ascorbate alone (•); ascorbate + 20% hematocrit (∆); ascorbate + 30% hematocrit (❑) ; ascorbate + 40% hematocrit (O). Traces presented as mean ± SD (n =3 ), but note that except at longer time points the errors are less than 2.5% of the mean, thus error bars are generally not visible within the figure. (c) Reduction of 100 µM MetHb-DBBF by 100 µM ascorbate in the presence of varying concentration of erythrocyte ghosts. Traces as per (b) except that ghosts were used rather than erythrocytes. Statistical analysis (see experimental) revealed that ascorbate significantly increased metHb reduction and this was enhanced by the addition of erythrocytes (but not ghosts). There was no difference between the effect of 20%, 30% or 40% hematocrit. (d) Synergistic effects of ascorbate and erythrocytes on the reduction of 100 µM MetHb-DBBF. Change in oxyHb-DBBF concentration with time. 40% hematocrit alone (); 100 µM ascorbate alone (•); 100 µM ascorbate + 40% hematocrit (O).

Figure 5

 5 Figure 5 Effect of ascorbate on hemoglobin radicals (a) EPR spectra 30s after addition of hydrogen peroxide (100 µM) to MetHb-DBBF (50 µM) in PBS at 37°C in presence or absence of sodium ascorbate (200 µM), added immediately prior to the peroxide. Ascorbate spectrum illustrated at 5x magnification. (b) As (a) but experiment measured at room temperature. (c) EPR spectrum of rabbit plasma following addition of Met-DBBF (50µM) and hydrogen peroxide (100 µM), sample was frozen 30s after peroxide addition. Overlaid with spectrum from (a) of peroxide addition to MetHb-DBBF in presence of ascorbate. Plasma spectrum illustrated at 5x magnification. (d) As (c), but plasma pre-incubated for 30 min. with ascorbate oxidase (15 units, Cucurbita sp., Sigma Chemical Co.). Any contributions of the metHb g = 1.99 signal to low temperature studies were removed by subtraction of appropriate concentrations of the pure metHb spectrum. EPR conditions (a, c, d): temperature = 10K, microwave power = 50 µW, microwave frequency = 9.47 GHz, modulation amplitude = 3G, time constant = 81.92 ms, scan rate = 3.6 G/s; (b): temperature = 295K, microwave frequency 9.62 GHz, microwave power 2 mW, modulation amplitude 1 G, scan rate 1.2 G/s; time constant 81.92 ms.

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341 Copyright 2006 Biochemical Society Packard

  8453) scanning from 450 to 700 nm with formation of oxyHb-DBBF and reduction of MetHb-DBBF monitored at 576 nm and 630 nm, respectively. MALDI-MS of the MetHb-DBBF samples post centrifugation could not detect any contamination from HbA o , indicating hemolysis was insignificant in the time course of the study.

	circulating water bath at 37°C under gentle agitation. Samples were evaluated at baseline
	and every 30 minutes for a maximum of 2 hours. Briefly, samples were centrifuged at
	6000 rpm for 2 minutes to separate the erythrocytes from MetHb-DBBF. The

hematocrit levels using pH 7.4 PBS buffer supplemented with 0.1% human serum albumin (ZBL) and 0.1% glucose (Sigma, St. Louis MO). 100 µM MetHb-DBBF and 100 µM ascorbate were then added. Immediately, thereafter all tubes were placed in a supernatant was diluted and measured in a photodiode array spectrophotometer (Hewlett

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341 Copyright 2006 Biochemical Society

	were run under the same conditions as the samples and subtracted to remove background
	signals from the cavity, prior to subsequent analysis. Quantitation of EPR signals in
	frozen blood was as described previously [29], using pure MetHb, ferric transferrin and
	Cu(II)-EDTA and as standards for g = 6, g = 4.3 and g = 2 signals respectively. In pure
	Hb solutions, separate quantitation of the Hb Tryptophan peroxyl radical from the Hb
	tyrosyl radical was performed by the technique of subtraction with a variable coefficient
	described previously [30], focusing initially on the g = 2.033 region where only the
	peroxyl radical contributes. The separate quantitation of radical species was not possible
	in whole blood due to the low radical concentrations and the presence of ceruloplasmin.
	Room temperature EPR measurements were performed using a Wilmad WG-813-TMS
	Aqueous Cell.
	The concentration of labile iron able to directly bind to transferrin in the Hb-DBBF
	solution was determined ex vivo. The iron bound to transferrin in venous plasma samples
	from one of the control experiments in rabbits was measured by EPR spectroscopy. These
	samples were then allowed to thaw. A solution of the Hb-DBBF was added to give a
	dilution of 20%, as in the in vivo experiment. The solutions were left for one hour at
	room temperature before being refrozen and the increase in the amount of iron bound to
	transferrin measured by EPR.
	Whole blood and plasma samples were transferred immediately
	into EPR tubes, frozen in methanol cooled in dry ice and placed in liquid nitrogen storage
	until analysis by EPR spectroscopy. All low temperature EPR spectra were measured at
	10K in a Bruker EMX spectrometer, with a spherical high quality Bruker resonator
	SP9703 and an Oxford Instruments liquid helium system. In all cases samples of water

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

  The principle, aqueous low molecular weight reducing agents in plasma are urate, glutathione and ascorbate. As implied by earlier work on HbA o ,[44] in vitro experiments failed to demonstrate any significant reduction of metHb-DBBF by glutathione or urate

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341 Copyright 2006 Biochemical Society animals

  . Thus the normal physiological defence mechanisms are able to prevent Hb-H formation following a 20% transfusion of Hb-DBBF, unlike the case when heme is released outside the erythrocyte in certain disease states e.g. rhabdomyolysis[13, 14],subarachnoid hemorraghe[15]. The chemical reactivity of ascorbate described here is therefore sufficient, in and of itself, to explain all of the plasma defences to heme-protein mediated oxidative stress.

	Biochemical

Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

  sufficient to generate 30 µM external ascorbate per hour[61]. It also explains why in the

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

	How does ascorbate compare to other plasma antioxidants (e.g. urate and glutathione) in
	dealing with heme-protein mediated oxidative stress? We propose three roles for
	ascorbate. Reduction of exogenous methemoglobin, reduction of ferryl and globin free
	radical scavenging. The reduction of methemoglobin cannot be catalysed by glutathione
	or urate and is unique to ascorbate. However, urate does have the potential to contribute
	to ferryl and protein radical scavenging. Urate is a good radical scavenger and can
	scavenge globin radicals [63]. Urate is also able to reduce ferryl [64]. The published rate
	constants however, suggest that at similar concentrations ascorbate outperforms urate
	[65]. We performed studies with ferrylHb-DBBF at the approximate concentrations
	present in rabbit plasma (50 µM ascorbate, 30 µM urate) and confirmed that ascorbate is
	a kinetically more competent ferryl reductant under these conditions (results not shown).
	One other reaction that likely contributes to ferryl reduction is electron transfer from
	ferrous hemoglobin [65] Although slower than reduction by either ascorbate or urate, the
	higher concentrations of deoxyHb-DBBF in vivo are likely to make this a major reaction

Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341

	(g)	TABLES AND FIGURES
			Time	Pre-	5	15	30	60	120	180
			(min. post	transfusion
			transfusion)	control
	Parameter
	MAP (mm Hg)		+ DBBF	80.8 ± 11.9	93.8 ± 9.2 ^91.7 ± 10.7	98.0 ± 13.0 *	94.4 ± 9.1*	88.1 ± 12.7	92.3 ± 7.7*
			Control	80.1 ±	81.1 ±	75.5 ±	78.3 ±	73.3 ±	71.9 ±	70.4 ±
				12.9	11.5	13.5	12.8	8.8	10.5	9.9
	Heart Rate (bpm)	+ DBBF	264.2 ± 21.5	234.2 ± 30.4 ^219.2 ± 41.5	232.5 ± 32.0	233.3 ± 22.3	226.0 ± 35.8	240.0 ± 34.5
			Control	249.3 ±	246.7 ±	248.3 ±	245.0 ±	237.5 ±	241.4 ±	229.3 ±
				25.2	25.2	27.3	32,7	25.6	26.1	28.2
	Blood pH		+ DBBF	7.38 ±	7.40 ±	7.40 ±	7.40 ±	7.41 ±	7.42 ±	7.43 ±
				0.04	0.03	0.04	0.04	0.05	0.03	0.04
			Control	7.37 ±	7.39 ±	7.39 ±	7.39 ±	7.39 ±	7.38 ±	7.40 ±
				0.06	0.03	0.02	0.03	0.03	0.02	0.03
	Hematocrit	+ DBBF	40.9 ± 3.6	31.5 ± 3.6 ^30.9 ± 3.6 *	30.5 ± 3.2 *	31.0 ± 3.5 *	29.8 ± 3.0 *	29.7 ± 3.0 *
			Control	37.6 ±	36.6 ±	37.0 ±	36.7 ±	37.0 ±	35.0 ±	35.0 ±
				3.0	2.8	2.3	2.2	1.9	2.2	2.4
	Total Hb (g/dl)	+ DBBF	12.5 ± 1.7	11.1 ± 1.0 ^10.9 ± 1.2	11.0 ± 1.2	11.1 ± 1.3	10.7 ± 1.0	10.5 ± 0.5
			Control	11.9 ±	11.4 ±	11.7 ±	11.7 ±	11.4 ±	11.2 ±	10.8 ±
				0.9	0.6	0.5	1.3	0.7	1.1	0.7
	Ceruloplasmin	+ DBBF	1.53 ±	1.48 ±	1.60 ±	1.73 ±	1.87 ±	1.71 ±	1.62 ±
	(µM)			1.27	0.91	1.32	1.45	1.57	1.75	1.60
			Control	2.47 ±	2.29 ±	2.37 ±	2.35 ±	2.37 ±	2.35 ±	2.41 ±
				1.81	1.71	1.84	1.81	1.71	1.81	1.75
	Transferrin Iron (µM)	+ DBBF	11.40 ± 2.03	17.14 ± 3.55 ^17.50 ± 3.43 *	18.16 ± 3.69 *	18.68 ± 3.56 *	20.03 ± 4.54 *	18.79 ± 5.66 *
			Control	10.74 ± 4.09	9.55 ± 3.01 ^9.47 ± 3.52	9.46 ± 3.59	9.23 ± 3.26	8.56 ± 2.71	8.08 ± 2.30
	Erythrocyte	+ DBBF	0.083 ±	0.064 ±	0.071 ±	0.089 ±	0.047 ±	0.058 ±	0.051 ±
	radical (µM)		0.061	0.026	0.033	0.031	0.026	0.062	0.046
			Control	0.053 ±	0.049 ±	0.041 ±	0.088 ±	0.046 ±	0.046 ±	0.045 ±
				0.012	0.017	0.007	0.075	0.015	0.011	0.018
	Met Hb (µM)	+ DBBF	3.38 ±	Trust Biomedical Research Collaboration 33.36 ± 29.09 ± 23.17 ± 15.17 ± 8.21 ±	6.48 ±
	1.23 Grant, (CEC, PM), a BBSRC studentship (JMD) and the European Union STREP, 9.75 ^# 8.08 * 8.11 * 7.00 * Control 2.69 ± 2.49 ± 2.56 ± 2.63 ± 2.41 ± 2.64 ± 3.02	2.95 2.61 ±
	"EUROBLOODSUBSTITUTES" (CEC, MTW, PM, RD). We thank Dr. Dimitri 1.05 0.82 0.80 0.90 0.71	0.55	0.81
	Svistunenko (University of Essex) for assistance with the interpretation of EPR spectra. Table 1 Physiological and EPR parameters following 20% transfusion with Hb-DBBF

Biochemical Journal

Biochemical Journal Immediate Publication. Published on 18 Jul 2006 as manuscript BJ20060341 Copyright 2006 Biochemical Society

(f)

Table 1 Legend Physiological and EPR data for animals undergoing control transfusions of autologous blood (n=7) and those undergoing transfusions with DBBF (n=6). All data mean ± SD. In the Hb-DBBF group, data were corrected for the blood removed by the drop in hematocrit. The methemoglobin concentration in the EPR samples was calculated by comparing the size of the strong high field feature (g = 6) to that of a pure methemoglobin standard of known concentration. To remove the problem of the low field