Irina Maluykova

Oksana Gutsal

Marina Laiko

Anne Kane

Mark Donowitz

Olga Kovbasnjuk

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells

Keywords: Shiga toxin, intestinal epithelial cells, transcytosis, actin turnover, latrunculin B

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic E. coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.

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M A N U S C R I P T

Introduction

Shiga toxins 1 and 2 (Stx1, Stx2), are major virulence factors in foodborne illnesses caused by enterohemorrhagic E. coli (EHEC). Stx1 and 2 belong to a class of so-called AB 5 bacterial toxins [START_REF] Acheson | The family of Shiga and Shiga-like toxins[END_REF], which consist of a single catalytic A-subunit and a pentamer of identical B-subunits, the latter responsible for receptor binding and intracellular trafficking. The A-subunit of Stx1

possesses N-glycosidase activity and inhibits protein synthesis through specific removal of the adenine base at position 4324 of the 28S ribosomal RNA of the 60S subunit of the eukaryotic ribosome.

Individuals who become infected with EHEC often develop watery diarrhea, which then becomes bloody, followed by recovery. However, up to 10% of infected individuals, mostly children, develop life-threatening systemic complications including hemolytic uremic syndrome (HUS) [START_REF] Acheson | The family of Shiga and Shiga-like toxins[END_REF][START_REF] Siegler | Hemolytic uremic syndrome; pathogenesis, treatment, and outcome[END_REF]. Antibiotic use against the EHEC infections has proven to be harmful, increasing the risks of HUS [START_REF] Tarr | Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome[END_REF][START_REF] Wong | The risk of the hemolyticuremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections[END_REF].

The principal gastrointestinal and systemic manifestations of Stx-related diseases are believed to be due to the action of the toxins on vascular endothelial cells, which express the glycosphingolipid Gb3, the only well characterized Stx receptor [START_REF] Donohue-Rolfe | Pathogenesis of Shigella diarrhea. 9. Simplified high-yield purification of shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies[END_REF][START_REF] Jacewicz | Pathogenesis of Shigella diarrhea.11. Isolation of a Shigella toxin-binding glycolipid from rabbit jejunum and Hela cells and its identification as globotriaosylceramide[END_REF]. EHEC strains are not generally invasive like the prototypic Stx-producing Shigella dysenteriae type 1. The question of how Stx gains access from the gut lumen to the underlying endothelial cells thus is crucial to understand the systemic complications of EHEC.

Stx1 might traverse intestinal epithelial layer by transcellular or paracellular pathways. It has been shown that Gb3 is absent from human intestinal epithelial cells [START_REF] Holgersson | Glycosphingolipids of the human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures[END_REF][START_REF] Ergonul | Shigatoxin-1 binding and receptor expression in human kidneys do not change with age[END_REF][START_REF] Schüller | Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture[END_REF][START_REF] Kovbasnjuk | The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer[END_REF][START_REF] Miyamoto | Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo[END_REF]. This excludes a

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Gb3-dependent route of toxin transcytosis and adds more complexity to the mechanism of interaction between the toxin and intestinal epithelial cells.

Several studies have been performed to elucidate the pathways of Stx transcytosis across intestinal epithelial cells. Translocation of Stx1 was examined [START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF][START_REF] Sherman | Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium[END_REF] in highly polarized intestinal epithelial CaCo-2A cells (Gb3 positive) and T84 cells (Gb3 negative). Biologically active Stx1 was translocated from the apical to the basolateral surfaces of both cell lines, which indicates that toxin transcytosis does not require Gb3 receptor binding. Stx1 transcytosis did not affect either transepithelial electrical resistance (TER) or movement of [3H]inulin, suggesting that apical-to-basolateral toxin movement was through the cells rather than the paracellular pathway [START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF][START_REF] Sherman | Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium[END_REF]. The T84 cells have been shown to be resistant to Stx1-induced cytotoxicity over 24h of exposure [START_REF] Schüller | Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture[END_REF][START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF][START_REF] Sherman | Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium[END_REF]. However, the efficiency of Stx1 transcytosis was relatively low and only ~ 2% of the apical amount of Stx1 was transferred across the cell monolayer in 24h.

We speculated that the process of transcellular toxin transcytosis might be facilitated in EHEC infection. During the course of infection, EHEC subvert the host cell signaling machinery to rearrange the actin cytoskeleton and produce characteristic attaching and effacing lesions on the surface of intestinal epithelial cells [START_REF] Goosney | Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals[END_REF][START_REF] Caron | Subversion of actin dynamics by EPEC and EHEC[END_REF]. This actin rearrangement might facilitate the toxin movement across the monolayer. Therefore, the goal of these studies was to determine the effect of actin polymerization-depolymerization on toxin transcytosis across the intestinal epithelial barrier.

Here amount of at least one actin-binding protein, such as phospho-cofilin-1. We suggest that changes in actin cytoskeleton similar to these caused by LatB might occur during EHEC interaction with the intestinal epithelium.

Materials and Methods

Materials

Purified Stx1 and recombinant Stx1B were prepared by Dr. A. Kane (GRASP Center, Tufts New England Medical Center, Boston, MA) as previously described [START_REF] Acheson | One step high yield affinity purification of Shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays[END_REF][START_REF] Acheson | Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems[END_REF]

Cell culture

Human colonic epithelial T84 cells (A.

Quantification of G-actin and F-actin

Fractionation of G-actin and F-actin was performed by Triton X-100 extraction of intracellular actin as described [START_REF] Ivanov | Differential roles for actin polymerization and a myosin II motor in assembly of the apithelial apical junctional complex[END_REF]. Briefly, T84 cells were washed with HBSS and G-actin was extracted by gentle shaking for 5 min at room temperature in HBSS containing 1% Triton X-100, proteinase inhibitor cocktail and 1 µg/ml phalloidin to prevent filament disassembly. The Triton X-100-soluble G-actin fraction was mixed with an equal volume of 2x SDS sample buffer and boiled. Cells were then briefly washed with HBSS and the Triton X-100-insoluble F-actin fraction was collected by scraping cells in two volumes of SDS sample buffer, and boiled. The amount of actin in each fraction was determined by gel electrophoresis, Western blotting, and Odyssey Fluorescence Imaging System as described above.

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Statistical analysis

Values are presented as mean ± SEM. Normal distribution for each dataset was tested using statistical package available at http://home.ubalt.edu/ntsbarsh/. Statistical significance was determined using Student's unpaired ttest and p-value <0.05 was considered significant.

Results

Stx1 and Stx1B are both transcytosed across T84 cells

It has been previously shown that ~ 2% of apically added active Stx1 holotoxin translocates across the T84 monolayer in 24h [START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF]. We repeated these experiments using both fluorescently As in case of holotoxin [START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF], Stx1B-680 transcytosis in T84 cells was also insensitive to 0.1µM BFA, indicating a lack of involvement of the retrograde pathway in toxin transcytosis.

Higher doses of BFA were toxic to the T84 cells, caused a significant drop in TER and apoptosis characterized by caspase-3 activation (data not shown).

Stx1 and Stx1B are internalized by Gb3 free intestinal epithelial T84 cells

Stx1 movement through the Gb3-negative T84 monolayers without changes in TER or paracellular permeability [START_REF] Schüller | Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture[END_REF][START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF] suggests that in the absence of Gb3 the Stx1 is endocytosed by

an as yet uncharacterized mechanism. We hypothesized, if Stx1/Stx1B uptake by Gb3-free cells is a receptor mediated process (possibly another low affinity or low expression receptor), then first order reaction kinetics should apply, which predicts a concentration and time-dependent saturated uptake due to receptor saturation [START_REF] Zorko | Cell-penetrating peptides: mechanism and kinetics of cargo delivery[END_REF]. To determine the toxin uptake as a function of

Cortical actin is involved in Stx1/Stx1B uptake by T84 cells

Fluid phase endocytosis is an actin-driven process and in polarized cells the cargo resides inside apical actin-coated vesicles [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF][START_REF] Mettlen | Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells[END_REF][START_REF] Jones | Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides[END_REF]. We found that during uptake, Stx1B resides inside spherical actin vesicles on the apical surface of T84 cells (Fig. 2A-F). Moreover, Stx1B uptake is accompanied by an increase in apical F-actin amount. However, inside the cells the Stx1B vesicles are not covered by actin (Fig. 2G), indicating that Stx1B-containing vesicles undergo some sub-apical sorting. We concluded that in receptor-free intestinal epithelial cells, actin remodeling is involved in Stx1/Stx1B uptake into some sub-apical compartment.

LatB significantly increased the transcytosis and uptake of Stx1B

Based on our data, we hypothesized that changes in actin cytoskeleton might facilitate toxin movement across the intestinal epithelial layer. Subsequently, the effect of actin polymerizingdepolymerizing agents, including CytD, LatB and Jasp, on Stx1/Stx1B uptake and transcytosis was studied. For these experiments, T84 cells were treated for 24h with Stx1B-680 and 1µM of Jasp or LatB were apically added for the last 4h or 1µM of CytD for the last 2 h. These treatments had been previously reported to significantly affect the F-actin pool in T84 cells [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF][START_REF] Ivanov | Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex[END_REF].

Quantification of Stx1B in total cell lysates (Fig. 3A) showed significant increase in the amount of intracellular toxin due to LatB. CytD significantly decreased the intracellular amount of Stx1B, while Jasp had no effect on the amount of intracellular toxin. Quantification of Stx1B in the lower chamber showed that LatB significantly increased toxin transcytosis, while neither CytD nor Jasp affected transcytosis (Fig. 3 B andC).

These data show that LatB treatment possibly increases both the toxin uptake across the apical membrane and toxin efflux through the basolateral membrane. To test whether actin modification by LatB stimulates both processes, the effect of LatB on Stx1B uptake and transcytosis was measured over shorter periods of time than 24h. For these experiments T84 cells were exposed simultaneously to 1 µg/ml Stx1B-680 and 1µM LatB over different times between

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compared to control cells incubated with Stx1B only (Fig. 4 A and B). Incubation with LatB over 30 min significantly increased the amount of intracellular toxin compared to control cells; 4h of LatB treatment increased the intracellular amount of Stx1B ~ 5 times. LatB also significantly increased Stx1B transcytosis, indicating that in T84 cells LatB facilitates the Stx1B transport across both apical and basolateral membranes. However, there was a time delay in LatB stimulated Stx1B transcytosis compared to its effect on Stx1B uptake, and at times shorter than 1h, LatB treatment did not affect transcytosis (Fig. 4B). Treatment of T84 cells with different concentrations of LatB over 4h showed significant increase of Stx1B transcytosis starting at 0.5 µM LatB (Fig. 4C).

It is well established that latrunculins, including LatB, significantly decrease the TER of polarized intestinal epithelial monolayers, including T84 cells [START_REF] Ivanov | Differential roles for actin polymerization and a myosin II motor in assembly of the apithelial apical junctional complex[END_REF][START_REF] Matthews | Effects of F-actin stabilization or disassembly on epithelial Cl-secretion and Na-K-2Cl cotransport[END_REF][START_REF] Shen | Actin depolymerization disrupts tight junctions via caveolaemediated endocytosis[END_REF]. To ensure that LatB induced increase in Stx1 transcytosis was not caused by changes in TER (Fig. 4 B andC) and possibly changes in paracellular permeability [START_REF] Jones | Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides[END_REF][START_REF] Ivanov | Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex[END_REF], but is due to the actin-dependent changes in toxin uptake and transcellular trafficking, T84 cells were treated basolaterally for 4h with 0.1% tannic acid, a cell-impermeable cross-linker of cell surface carbohydrates that selectively fixes the plasma membrane but does not diffuse across the TJ [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF][START_REF] Newman | Effect of neuromimetics upon the release of atrial natriuretic peptide granules: are multiple pathways involved in secretion?[END_REF][START_REF] Polishchuk | Delivery of raft-associated, GPIanchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway[END_REF][START_REF] Paladino | GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells[END_REF]. This treatment did not change the properties of the TJ in T84 monolayers [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF]. Tannic acid completely inhibited the effect of LatB on Stx1B transcytosis (Fig. 4C), suggesting that LatB facilitated transcytosis of Stx1B is a transcellular process and actin polymerization-depolymerization plays an important role in its regulation. Basolateral tannic acid treatment in the presence of LatB also significantly increased the Stx1B amount in total cell lysates compared to non-treated controls. Thus, relative fluorescence intensity in control cells exposed to Stx1B-680 for 4h was 57 ± 11 units, while in cells treated with for 4h with 1µM LatB apically and tannic acid basolaterally the Stx1B-680

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relative intensity was 678± 37 units (p<0.05 vs. control, 3 independent experiments). Tannic acid alone did not affect the amount of intracellular toxin compared to the control cells, and Stx1B-680 relative intensity in the presence of tannic acid was 66 ± 8 units (3 independent experiments).

The LatB-mediated changes in cytoskeleton that facilitate Stx1B uptake and transcytosis through T84 monolayers are different from these leading to decrease in TER (Fig. 3), which occur rapidly 10-15 min after treatment and at low LatB concentrations that do not affect Stx1B

transcytosis [START_REF] Shen | Actin depolymerization disrupts tight junctions via caveolaemediated endocytosis[END_REF].

LatB facilitates the transcytosis of other molecules across intestinal epithelial cells

The effect of LatB on transcellular transcytosis has not been studied in detail in polarized

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LatB-induced change in cofilin-1 phosphorylation may be involved in Stx1B facilitated transcytosis

Additionally to the sequestration of monomeric G-actin, both Lat A and B has been shown to influence the expression or phosphorylation of several actin binding proteins [START_REF] Minamide | Differential regulation of actin depolymerizing factor and cofilin in response to alterations in the actin monomer pool[END_REF][START_REF] Quinlan | Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways[END_REF]. We tested whether LatB-induced increase in G-actin is also accompanied by changes in the expression or postranslational modification of some G-actin-binding proteins in T84 cells, such as cofilin and profilin [START_REF] Yarmola | Profilin: emerging concepts and lingering misconceptions[END_REF][START_REF] Ono | Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics[END_REF]. For these experiments, cells were treated with 1µM LatB over different time intervals between 15 min and 4h, and the expression of cofilin-1 and its phosphorylated form, and profilin, were tested in total cell lysates by immunoblotting. LatB treatment significantly increased the cofilin-1 phosphorylation, which occurred as early as 15 min and continued over 4h (Fig. 5). Moreover, the increase in the amount of phospho-cofilin-1 over time of LatB treatment correlates (r= 0.93 by correlation test for independent variables from Excel) with increase in the amount of G-actin, reflected in G/F actin ratio (Fig. 5C). Neither the total amount of cofilin-1 nor profilin changed over these experimental time intervals (Fig. 5). Additionally, the total amount of other actin-binding proteins such as ezrin and vinculin did not change upon LatB treatment (data not shown). These data suggest that facilitation of transcellular transcytosis by LatB may be related to LatB-induced changes in the activity of cofilin-1.

Discussion

The Stx translocation across the intestinal epithelial layer to spread systemically is a necessary step in the development of the EHEC-induced spectrum of disease. However, the mechanisms of this translocation are unknown. The ability of fully active Stx1 and Stx2 to cross the epithelial barrier through the cells has been demonstrated [START_REF] Acheson | Translocation of Shiga toxin across polarized intestinal cells in tissue culture[END_REF][START_REF] Sherman | Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium[END_REF][START_REF] Hurley | Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells[END_REF][START_REF] Hurley | Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration[END_REF][START_REF] Di Pasquale | AAV transcytosis through barrier epithelia and endothelium[END_REF]. However, the

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efficiency of transcytosis was very low with only ~ 2% of apical Stx1 crossing monolayers in 24h. Although such a small amount of toxin potentially could be responsible for systemic complications, we speculated that the changes in host cell protein expression and particularly, in actin and actin-binding proteins due to EHEC attachment and effacement [START_REF] Goosney | Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals[END_REF][START_REF] Caron | Subversion of actin dynamics by EPEC and EHEC[END_REF] might stimulate toxin uptake and transcytosis. The impact of EHEC infection on the host proteins has not been investigated in detail. However, there are some evidences that EHEC colonization changes the protein expression in intestinal epithelial cells [START_REF] Robinson | Shiga toxin of enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal colonization[END_REF]. Moreover, proteomic analysis of intestinal epithelial Caco-2 cells infected with enteropathogenic E.coli (EPEC), which form attaching/effacing lesions similar to those of EHEC, showed significant increase in the abundance of multiple actin depolymerizing proteins [START_REF] Hardwidge | Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli[END_REF]. For instance, actin severing proteins gelsolin and cofilin were upregulated 3.57 and 2.5 times, respectively. This shows that changes in actin binding proteins occur as a part of bacterial attachment and effacement to the intestinal epithelial cells.

Here we demonstrate that actin depolymerizing drug LatB significantly increases Stx1B transcytosis in a time and a concentration-dependent manner. It is known that LatB decreases TER and increases permeability to ions and uncharged molecules with size < 10kDa [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF][START_REF] Shen | Actin depolymerization disrupts tight junctions via caveolaemediated endocytosis[END_REF]. We show that the LatB facilitated Stx1B transcytosis is not across the TJ, because Stx1B transcytosis in the presence of LatB is completely blocked by basolateral application of tannic acid. Recently the addition of tannic acid to the basolatheral cell surface has been shown to inhibit the apical to basolateral transcellular transcytosis of adeno-associated virus [START_REF] Di Pasquale | AAV transcytosis through barrier epithelia and endothelium[END_REF]. The selectivity of tannic acid (apical vs. basolateral) has been previously tested in T84 cells [START_REF] Utech | Mechanism of IFN-γ-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane[END_REF], and apically (but not basolaterally) applied tannic acid completely prevented cholera toxin apical uptake. In contrast,

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basolateral tannic acid (but not apical) effectively inhibited uptake of transferrin from the basolateral side. Taken together, our data show that LatB facilitates Stx1B transepithelial flux is transcellular and not paracellular. Additionally, both CytD and Jasp treatments have also been shown to decrease TER of intestinal epithelial monolayers [START_REF] Matthews | Effects of F-actin stabilization or disassembly on epithelial Cl-secretion and Na-K-2Cl cotransport[END_REF][START_REF] Mark | Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption[END_REF][START_REF] Shurety | Fluid-phase markers in the basolateral endocytic pathway accumulate in response to the actin assembly-promoting drug jasplakinolide[END_REF]. However, neither drug increased Stx1B transcytosis in T84 cells, supporting our conclusion that LatB facilitates transcellular transcytosis of toxin. The increase in Stx1B amount in total cell lysates due to the tannic acid treatment in the presence of LatB additionally supports that the toxin moves across the cells.

It has been shown that CytD inhibits both receptor mediated and fluid phase endocytosis at the apical surface [START_REF] Lamaze | The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells[END_REF][START_REF] Gottlieb | Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells[END_REF][START_REF] Jackman | Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D[END_REF], while Jasp stimulates basolateral fluid phase endocytosis, but does not affect apical uptake [START_REF] Shurety | Fluid-phase markers in the basolateral endocytic pathway accumulate in response to the actin assembly-promoting drug jasplakinolide[END_REF]. These data are in good agreements with our findings that CytD inhibits Stx1B apical uptake, and Jasp does not change it. In general, latrunculins significantly inhibit receptor mediated endocytosis [START_REF] Lamaze | The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells[END_REF][START_REF] Volovyk | Agonist-stimulated betaadrenergic receptor internalization requires dynamic cytoskeletal actin turnover[END_REF]. However, latrunculins have been shown to stimulate internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the cystic fibrosis transmembrane conductance regulator (CFTR), while the mechanisms of endocytosis have not be addressed [START_REF] Ganeshan | CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization[END_REF][START_REF] Zhou | Contribution of cytoskeleton to the internalization of AMPA receptors[END_REF]. The increase in apical Stx1B uptake by LatB (but not by CytD or Jasp) serves as additional evidence that Gb3-free T84 cells take up Stx1B by a nonreceptor mediated pathway.

These results prompted us to look for the LatB induced molecular changes which might be involved in the regulation of transcellular transcytosis. LatB affects the kinetics of actin polymerization by forming 1:1 molar complexes with G-actin [START_REF] Coue | Inhibition of actin polymerization by latrunculin A[END_REF][START_REF] Spector | Latrunculins-novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D[END_REF]. The actin sequestration over time leads to changes in activity and expression of several actin binding proteins, including vinculin, VASP, profilin, cofilin and ADF [START_REF] Minamide | Differential regulation of actin depolymerizing factor and cofilin in response to alterations in the actin monomer pool[END_REF][START_REF] Quinlan | Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways[END_REF]. The novelty in our experiments is that LatB

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rapidly (15 min) increased the amount of phosphorylated cofilin-1, which is inactive in terms of actin binding [START_REF] Huang | Cofilin phosphatases and regulation of actin dynamics[END_REF], while the total amount of this protein was unchanged. This effect was sustained over the 4h of experimental time. Cofilin-1 is a ubiquitous mediator of actin filament turnover in eukaryotes. In the phosphorylated form cofilin is unable to bind to actin, while the dephosphorylation of cofilin enables its actin severing and depolymerizing activity [START_REF] Minamide | Differential regulation of actin depolymerizing factor and cofilin in response to alterations in the actin monomer pool[END_REF][START_REF] Huang | Cofilin phosphatases and regulation of actin dynamics[END_REF].

LatB-induced cofilin-1 phosphorylation should lead to F-actin destabilization and this might be important for stimulation of the transcellular pathway. A possibility that LatB influences the expression of other proteins involved in actin turnover in intestinal epithelial cells remains to be tested.

Interestingly, this LatB mediated route for transcytosis might be a common pathway for many molecules which are able to traffic across epithelial monolayers. It has been previously

shown that cholera toxin (CT) and its B-subunit are transcytosed transcellularly by T84 cells, and BFA did not inhibit, but rather stimulated this process [START_REF] Lencer | Transcytosis of cholera toxin subunits across model human intestinal epithelia[END_REF]. Moreover, CT transcytosis directly correlated with CT-induced Cl-secretion, indicating functional importance of this pathway. The results allow us to speculate that interaction of bacteria or bacterial products with intestinal epithelial cells might potentially recreate the LatB-induced molecular changes which significantly facilitate the transcellular transcytosis of toxins and other antigens. These data that two different AB5 toxins [START_REF] Sandvig | Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin[END_REF] move across the intestinal epithelial cells by a Golgi/ER independent pathway shows the importance of transcellular transcytosis in the interactions of intestinal epithelial cells with a broad variety of bacterial products, which similarly to Stx might gain access to the underlying tissues.

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  using an in vitro intestinal epithelial T84 cell model, we demonstrated that latrunculin B (LatB), significantly increases the transcytosis of the Stx1 B-subunit (Stx1B). Complete inhibition of LatB-induced Stx1B transcytosis by basolateral application of tannic acid demonstrates that this toxin movement occurs through the cells, rather than through tight A C C E P T E D M A N U S C R I P T junctions (TJ). The effect of LatB on Stx1 transcytosis is specific, and neither cytochalasin D (CytD) nor jasplakinolide (Jasp), which both affect filamentous actin turnover and similarly to LatB decrease TER of intestinal epithelial monolayers, did not influence Stx1B transport across the T84 cells. This LatB-facilitated Stx1B transcellular transcytosis is associated with changes in

  . The quality of Stx1 and Stx1B has been checked on 15% SDS-PAGE followed by GelCode Blue staining (Pierce, Rockford, IL) and two bands (~ 32kDa for A-subunit and) or one band (< 10kDa for B-subunit) respectively, were detected. B-subunit of Cholera toxin (CTB) was from List Biological Laboratories, Inc. LatB was from Axxora (San Diego, CA). The following antibodies were rabbit polyclonals: anti-cofilin, a generous gift from Dr. J.R.Bamburg (Colorado State University, CO), anti-phospho-cofilin and anti-profilin from Cell Signaling Technology (Danvers, MA). Rabbit monoclonal anti-GAPDH was from US Biological (Swampscott, MA), mouse monoclonal anticathepsin D was a gift from Dr. A. Hubbard (Johns Hopkins University, Baltimore, MD). All fluorescent secondary antibodies, phalloidin, and 40kDa dextran conjugated to Alexa 488 fluorescent dye were from Molecular Probes-Invitrogen (Carslbad, CA). Stx1, CTB, and recombinant Stx1B were conjugated with Alexa fluor 680, Alexa fluor 568, or Alexa fluor 488 A C C E P T E D M A N U S C R I P T reactive dyes according to the manufacturer's protocol (Molecular Probes-Invitrogen). All other chemical reagents were purchased from Sigma (St. Louis, MO).

A

  C C E P T E D M A N U S C R I P Tlocalization experiments, cells were incubated over the indicated time with 0.5 µg/ml Stx1B-568 and 0.5 µg/ml CTB conjugated to Alexa fluor 488 (CTB-488) and fixed as above. For Stx1B and lysosomal co-localization, cells were incubated over the indicated time with 0.5 µg/ml Stx1B-568, fixed, permeabilized and blocked as above, incubated with anti-cathepsin D antibody at room temperature for 1 h, washed 3 times in PBS and incubated with secondary fluorescent antibodies for an additional 1h. All cell preparations were mounted on glass slides for confocal microscopy examination. Fluorescent image acquisition of tissue was performed using a Zeiss 410 LSM confocal imaging system. Collected 8-bit fluorescence images of confocal optical sectioning with 0.5 μm steps were stored on disc and fluorescence intensity was quantified by MetaMorph software (Molecular Devices Corporation, Downingtown, PA), as we previously described[START_REF] Kovbasnjuk | Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells[END_REF].

  labeled holotoxin (Stx1-680) and B-subunit of Stx1 (Stx1B-680). Confluent T84 cells were exposed apically either to Stx1B-680 (0.1 µg), or to increasing amount of Stx1B-680 (0.06 µg, 0.125 µg, 0.25 µg and 0.5µg) in 0.5 ml of apical media. The relative amount of transcytosed Stx1-680 or Stx1B-680 was measured in the lower chamber media 24 h later by quantifying the toxin 680 nm relative fluorescence intensity. The absolute amount of transcytosed Stx1-680 or Stx1B-680 was calculated from fluorescence intensity by comparison to the Stx1-680 or Stx1B-680 fluorescence intensity calibration curve generated from three different concentrations of Stx1-680 or seven different concentrations of Stx1B in a range between 1µg/ml and 5ng/ml. Similarly to the non-fluorescent holotoxin [12], Stx1-680 and Stx1B-680 transcytosis across confluent T84 cells is a slow process. Only ~ 4 ng of Stx1-680, which corresponds to 3.9 ±0.6 % of total apical Stx1-680 (n= 6 filters) was transcytosed across the monolayer in 24h. In case of Stx1B, only ~ 4 ng, ~ 8ng, ~ 13ng and ~ 28ng of toxin respectively was recovered from lower chamber in 24h, which corresponds to 6.0 % ± 0.4 of total apical amount of toxin (n= 6 filters per each conditions). A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10 We also measured the TER of T84 monolayers treated for 24h with Stx1B-680 to ensure that there were no Stx1B-induced changes in the TJ. Similarly to the previously studied holotoxin [9,12,13] neither 1µg/ml of apical non-fluorescent Stx1B nor Stx1B-680 change TER over 24h of incubation. From these experiments we concluded that Stx1B is sufficient for holotoxin transcytosis across T84 monolayer and also that Stx1B-680 is a suitable tool to study the Stx1 translocation across the intestinal epithelial monolayer.

  the apical toxin concentration and as a function of time, T84 cells were incubated with Stx1-680 holotoxin or Stx1B-680 and the Stx1-680 or Stx1B-680 fluorescence intensity in total cell lysates, which corresponds to the relative amount of endocytosed toxin, was measured. Both, dose and time dependencies of toxin uptake demonstrated a linear non-saturating relationship between the amounts of endocytosed holotoxin or Stx1B and apical toxin concentration or A C C E P T E D M A N U S C R I P T incubation time, respectively (Fig. 1A and B). Additionally, incubation of T84 cells at 4ºC with fluorescently labeled Stx1B over 3h did not result in detectable toxin binding to the apical surface (data not shown). These results demonstrate that Stx1 and Stx1B uptake by T84 cells is not a receptor mediated process. To further test the hypothesis that Stx1/Stx1B uptake into T84 cells is mainly through non-receptor mediated endocytosis, T84 cells were incubated simultaneously with fluorescently labeled Stx1B and 40 kDa dextran, the latter being a fluid phase endocytosis marker, the MW of which closely matches the MW of the Stx1B pentamer. The distribution of Stx1B relative to the dextran was compared by confocal microscopy after 4 and 24h of co-incubation (Fig. 1C-F). The co-localization function (MetaMorph), showed that ~ 78±13 % of Stx1B intracellular vesicles were co-localized with 40kDa dextran vesicles upon 24h of co-incubation. This suggests that fluid phase endocytosis may be a major pathway for Stx1 uptake in receptor-free cells. The Stx1B co-localized significantly neither with B-subunit of cholera toxin (CTB) (Fig. 1G-I), which upon uptake by G M1 -receptor mediated endocytosis marks the whole retrograde pathway [21], nor with a marker for recycling endosomes, Rab11 (data not shown). However, the Stx1B intracellular pattern partially matched the pattern of the lysosomal marker cathepsin D (Fig. 1J-L).

  intestinal epithelial cells. Subsequently, we tested whether LatB stimulates the transcytosis of several molecules which use different endocytic mechanisms for uptake. For these experiments, T84 cells were incubated for 4h in the presence or the absence of 1µM LatB either with 40kDa dextran or CTB, the last binds to apical G M1 receptors. CTB can be taken up by clathrin-and caveolin-dependent and independent mechanisms and is transcytosed independently of its retrograde trafficking pathway[START_REF] Chinnapen | Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER[END_REF][START_REF] Lencer | Transcytosis of cholera toxin subunits across model human intestinal epithelia[END_REF][START_REF] Kirkham | Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles[END_REF]. Similarly to Stx1B, LatB increased dextran transcytosis from 19.5±4.2 relative fluorescent intensity units in control cells to 182.7±3.7 in the presence of LatB (p<0.05; n= 8 experiments). LatB also significantly increased the transcytosis of CTB from 30.2±2.8 relative fluorescent intensity units in control cells to 664.8 ±111.0 in the presence of LatB (p<0.05; n= 8 experiments).

Fig. 1 .

 1 Fig. 1. Stx1B uptake by T84 cells is not receptor-mediated, but is an actin-dependent process. A

Fig. 2 .Fig. 3 .

 23 Fig. 2. A -representative confocal image of the apical area of T84 cells which are in the process

Fig. 4 .

 4 Fig. 4. LatB time and dose effects on Stx1B uptake and transcytosis. A-Stx1B-680 relative

Fig. 5 .

 5 Fig. 5. Effect of LatB on expression of actin and actin-binding proteins. A -Representative

Acknowledgments

This work was supported by NIH grants RO1DK58928, R24DK064388 (The Hopkins Basic

Research Digestive Disease Development Core Center) and P30DK 34928 (the GRASP Center).

We acknowledge the editorial assistance of Ms. H. McCann.

A C C E P T E D

M A N U S C R I P T


G Jayakumar Amirtharaj

Sathish Kumar Natarajan

Ashis Mukhopadhya

George Zachariah

Sudheer K Hegde

George Kurian

K A Balasubramanian

Anup Ramachandran

Fatty acids influence binding of cobalt to serum albumin in patients with fatty liver Subject area of the article: Cell Biochemistry

Keywords: Fatty liver, oxidative stress, albumin, cobalt, fatty acids

Human serum albumin binds ligands such as fatty acids and metals in circulation.

Oxidative stress can modify albumin and affect ligand binding. This study examines the role of oxidative stress and fatty acids in modulating cobalt binding to albumin in patients with fatty liver. Elevated levels of malondialdehyde and protein carbonyls, indicative of oxidative stress was evident in serum of patients with fatty liver. A significant decrease in albumin-cobalt binding was also observed. Albumin isolated from patient serum also showed an increase in bound fatty acids. In vitro experiments indicated that while oxidant exposure or removal of fatty acids independently decreased cobalt binding to albumin, removal of fatty acids from the protein prior to oxidant exposure did not influence the oxidant effect on albumin-cobalt binding. These results suggest that oxidative stress & fatty acids on albumin can influence albumin-cobalt binding in patients with fatty liver by independent mechanisms.

anuprama@yahoo.co.uk, anup@cmcvellore.ac.in

A C C E P T E D M A N U S C R I P T

INTRODUCTION

Human serum albumin (HSA) is synthesized in the liver and is the most abundant protein in blood. It has a single polypeptide chain of 585 amino acids (66,500 Da) with three domains-I, II & III [START_REF] He | Atomic structure and chemistry of human serum albumin[END_REF]. Each domain consists of two sub domains A and B. The structure of albumin is stabilized by 17 disulphide bridges with a free thiol group at cysteine 34 which is prone to be oxidized [START_REF] Kawakami | Identification and characterization of oxidized human serum albumin. A slight structural change impairs its ligand-binding and antioxidant functions[END_REF]. Apart from maintaining colloid osmotic pressure, albumin also plays a role in transport of a number of ligands including hormones, hemin, bilirubin and fatty acids, as well as transition metals such as copper, cobalt, nickel, and cadmium [START_REF] Sadler | Involvement of a lysine residue in the Nterminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+[END_REF][START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF]. Modifications on albumin have been demonstrated in numerous pathophysiological conditions such as acute coronary syndrome [START_REF] Christenson | Characteristics of a n Albumin Cobalt Binding Test for assessment of acute coronary syndrome patients: a multicenter study[END_REF] and skeletal muscle ischemia [START_REF] Zapico-Muniz | Ischemia-modified albumin during skeletal muscle ischemia[END_REF]. These modifications can affect the protein's ability to bind ligands, as seen in chronic renal and liver disease [START_REF] Ivanov | Chronic liver and renal diseases differently affect structure of human serum albumin[END_REF]. It has been demonstrated that the binding of albumin to cobalt is compromised in patients with myocardial infarction [START_REF] Bhagavan | Evaluation of human serum albumin cobalt binding assay for the assessment of myocardial ischemia and myocardial infarction[END_REF] and myocardial ischemia [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF] and this forms the basis for the albumin-cobalt binding test (ACB) a marker for myocardial ischemia [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF].

The binding of metals to albumin has been well studied, and four different metal binding sites are known; site A, site B, the N-terminal and cys-34 [START_REF] Mothes | Evidence that the Principal Co(II)-Binding Site in Human Serum Albumin Is Not at the N-Terminus: Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia[END_REF]. Among the metals, binding is best characterized for copper and nickel [START_REF] Sadler | Involvement of a lysine residue in the Nterminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+[END_REF][START_REF] Laussac | Characterization of the copper(II)-and nickel(II)transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1 -24 of human serum albumin by 13C and 1H NMR spectroscopy[END_REF], which bind to the three Nterminal amino acid residues Asp-Ala-His. Cobalt is transported in circulation by albumin [START_REF] Sadler | Involvement of a lysine residue in the Nterminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+[END_REF] and earlier studies using N-terminal peptides of albumin indicated that the metal bound to the N-terminal at the sequence Asp-Ala-His-Lys (DAHK) [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF], similar to copper and nickel [START_REF] Sadler | Involvement of a lysine residue in the Nterminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+[END_REF]. However, a recent study has demonstrated that cobalt also binds to site A and B on the albumin molecule [START_REF] Mothes | Evidence that the Principal Co(II)-Binding Site in Human Serum Albumin Is Not at the N-Terminus: Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia[END_REF].

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Another important ligand for albumin is fatty acids and HSA is the primary transporter for the delivery of fatty acids to tissues, having at least seven binding sites for this ligand [START_REF] Simard | Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy[END_REF]. Oxidative stress is one of the mechanisms by which modification of albumin can occur [START_REF] Ivanov | Chronic liver and renal diseases differently affect structure of human serum albumin[END_REF], and free radicals have been implicated in the etiology of liver disease, playing a role in progression from fatty liver to fibrosis and cirrhosis [START_REF] Donnelly | Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease[END_REF][START_REF] Day | Steatohepatitis: a tale of two "hits"?[END_REF].

Since albumin is synthesized in the liver, and oxidative stress is known to be a feature of liver disease, we hypothesized that oxidative stress during fatty liver disease can cause modification on serum albumin, which may influence ligand binding properties of the protein. We tested this hypothesis by studying serum from patients with fatty liver and demonstrate that association of albumin with fatty acids is altered in an oxidative milieu and this can influence the cobalt binding capacity of the protein. All other chemicals and solvents used were of analytical grade.

METHODS

Tris

Patients and control subjects:

The study included 28 patients who were diagnosed with fatty liver by radiological findings, with high levels of alanine aminotransferase (ALT) and no history of alcohol consumption, diabetes mellitus or hyperlipoproteinaemia.

Controls were 28 patients with irritable bowel syndrome and no liver involvement. The following blood investigations were done in all subjects-aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, alkaline phosphatase (ALP), Oxidative stress parameters: Serum was used for the assessment of oxidative stress parameters. Protein carbonylation was determined spectrophotometrically based on the reaction of 2,4-dinitro-phenylhydrazine with protein carbonyls [START_REF] Sohal | Protein oxidative damage is associated with life expectancy of houseflies[END_REF] and the results were expressed as nmoles carbonyl content/ml of serum. Malondialdehyde, the end product of lipid peroxidation was measured by the thiobarbituric acid method using 1,1,3,3 tetramethoxy propane as standard [START_REF] Ohkawa | Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction[END_REF].

Assessment of cobalt binding to albumin:

The serum samples were assessed for cobalt binding as described with slight modifications [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF] treatment, the cobalt binding capacity was estimated as mentioned above. To confirm roles, if any, of hydrogen peroxide or superoxide in these changes, experiments were also carried out in the presence and absence of catalase (1000 units) or superoxide dismutase (SOD) (1000 units) respectively. To evaluate the role of hydroxyl radicals in the process, experiments were done with and without thiourea (50mM) and mannitol (50 mM), which have been used to scavenge hydroxyl radicals [START_REF] Grady | Hydroxyl radical production during oxidative deposition of iron in ferritin[END_REF] .

Lipid analysis from albumin: Total lipids were extracted from albumin as described [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF]. The lower organic phase was concentrated using nitrogen, resuspended in a small volume of chloroform/methanol (2:1) and used for lipid analysis. Neutral lipids were separated by thin layer chromatography (TLC) on silica gel G plates using the solvent system of hexane/diethyl ether/acetic acid (80:20:1, by vol). Spots corresponding to standard fatty acids run in parallel were identified by iodine exposure and eluted. The eluted fatty acids were methylated with methanol and concentrated HCl for 4 hours at 70°C, following which the fatty acid methyl esters were extracted with 1ml of heptane.

Individual fatty acid content was then quantitated using gas chromatography as described [START_REF] Cohen | Comparison of phospholipid and fatty acid composition of human erythrocytes and platelets[END_REF].

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Removal of fatty acid from purified HSA: Albumin (5.0 g /dL) was mixed with activated charcoal (2.5 g) and the pH of the solution was lowered to 3.0 by the addition of 0.2 N HCl.

The solution was then placed in an ice bath and mixed for 1 hour, following which the charcoal was removed by centrifugation at 3,000 rpm and the solution pH adjusted back to 7.0 by addition of 0.2 N HCl. Control experiments were carried out to confirm that changes in pH did not influence the cobalt binding assay.

Estimation of albumin: Albumin content was measured using the bromocresol green (BCG) method [START_REF] Doumas | Albumin standards and the measurement of serum albumin with bromcresol green[END_REF] .

Statistical

RESULTS

Patients with fatty liver on ultrasound, who were recruited for the study, had a significantly higher BMI when compared to controls (Table 1). . Serum levels of alanine transaminase were also significantly higher in patients when compared to controls, as were serum triglyceride levels. No difference in levels of gamma-glutamyltransferase was seen between controls and patients (data not shown). Interestingly, there was no significant difference in levels of serum albumin between patients and controls.

Initial experiments were then carried out to evaluate oxidative stress in serum from patients with fatty liver and the results are shown in Figure 1. Total protein oxidation assessed by protein carbonyls was significantly increased (P<0.05) in serum of

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patients with fatty liver (Figure 1A). This was accompanied by a significant increase in malondialdehyde (P<0.05) (Figure 1B) compared to control subjects.

Since albumin is the predominant protein in serum and has been shown to be susceptible to oxidative modification, it is possible that the increase in protein carbonyl content could reflect modification on the protein. To determine if oxidative stress in serum had any functional significance on albumin, we investigated the cobalt binding capacity of serum albumin in patients with fatty liver. As seen in figure 2, a slight but significant decrease in cobalt binding was seen in serum from patients with fatty liver, when compared to controls.

To determine whether oxidative stress could have an effect on albumin-cobalt binding, in vitro studies were then carried out using normal serum as well as purified human serum albumin. Exposure to hydrogen peroxide and copper sulfate resulted in a decrease in cobalt binding capacity of albumin on pure HSA (Figure 3A). Hydrogen peroxide alone had no effect, while copper sulfate alone showed a slight decrease which was not statistically significant. A similar result was obtained in serum as well (Figure 3B) where the decrease was prevented by presence of catalase (Figure 3B), confirming the role of hydrogen peroxide in the process.

Experiments were also conducted using other physiologically relevant oxidant generating systems such as the xanthine/xanthine oxidase system, which generates superoxide radicals. Surprisingly cobalt binding to albumin was not altered in presence of xanthine/xanthine oxidase (Figure 4A). This suggests that the superoxide anion per se does not influence albumin cobalt binding. However, the superoxide anion can be converted to hydrogen peroxide by superoxide dismutase. To confirm the results with xanthine/xanthine oxidase + superoxide dismutase, which would produce hydrogen peroxide alone, had no effect on albumin cobalt binding (Figure 4A). However, inclusion of copper sulfate resulted in a significant decrease in albumin cobalt binding, an effect which was reversed by addition of catalase (Figure 4A). All these data suggest that only hydroxyl radicals, produced by the Fenton reaction when hydrogen peroxide and copper are present could result in decreasing cobalt binding to albumin. To confirm this, experiments were repeated in presence of thiourea and mannitol, which can scavenge hydroxyl radicals [START_REF] Grady | Hydroxyl radical production during oxidative deposition of iron in ferritin[END_REF]. As seen in figure 4B, presence of thiourea and mannitol were able to significantly reverse the effect of hydrogen peroxide & copper on albumin cobalt binding, confirming the role of the hydroxyl radical in the process.

Albumin in circulation also binds to fatty acids and patients with fatty liver have been shown to have increased serum levels of fatty acids [START_REF] Lewis | Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes[END_REF]. To determine if the fatty acid content on albumin could influence the protein's binding to cobalt, blue sepharose was used to extract serum albumin from controls and fatty liver patients. The blue sepharose extraction resulted in isolation of serum albumin from both controls as well as fatty liver patients (data not shown). Fatty acid analysis from this albumin indicated that levels of oleic, myristic and lauric acid were increased in patients with fatty liver when compared to controls (Figure 5). These were the only fatty acids which were consistently detected as being associated with albumin from patients with fatty liver and controls.

A C C E P T E D M A N U S C R I P T

But can this increase in fatty acids influence cobalt binding to albumin? To examine this, in vitro experiments were carried out where fatty acids were removed from purified human serum albumin and the cobalt binding capacity of the samples analyzed.

Fatty acid removal from albumin was confirmed by TLC (data not shown). Interestingly, as seen in figure 6, cobalt binding to albumin decreased in samples where fatty acid had been removed, suggesting that fatty acid binding could influence cobalt binding to albumin.

The results so far suggest that oxidative stress is present in serum from patients with fatty liver, accompanied by a decrease in cobalt binding to albumin. In vitro exposure to free radicals or removal of fatty acids also decreases cobalt binding on albumin, suggesting that patients with fatty liver may have decreased fatty acids bound on albumin. Interestingly, however; analysis of albumin from patients showed an increase in fatty acids bound to albumin. This indicates that fatty acids may have a protective effect on maintaining albumin-cobalt binding. However, would removal of fatty acids aggravate the effect of oxidative stress on albumin-cobalt binding? This was tested in vitro where purified human serum albumin was subjected to oxidative stress subsequent to removal of fatty acids from the protein. As seen in figure 6, removal of fatty acids did not exacerbate the decrease in albumin-cobalt binding induced by oxidative stress, suggesting that oxidative stress and fatty acids influence albumin-cobalt binding by separate mechanisms.

DISCUSSION

Albumin, the predominant protein in circulation is mainly synthesized in the liver and binds to a number of ligands; serving as transport vehicle for numerous endogenous

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11 compounds including bilirubin, hemin, heavy metals and fatty acids [START_REF] Stewart | Interdomain zinc site on human albumin[END_REF]. The protein is known to be affected in conditions such as exposure to cigarette smoke [START_REF] Reznick | MANUSCRIPT 19 cigarette smoke as measured by protein carbonyl formation[END_REF][START_REF] Fabisiak | Quantification of oxidative/nitrosative modification of CYS(34) in human serum albumin using a fluorescence-based SDS-PAGE assay[END_REF], diabetes and renal diseases [START_REF] Toyokuni | Serum 4 -hydroxy-2-nonenal-modified albumin is elevated in patients with type 2 diabetes mellitus[END_REF][START_REF] Himmelfarb | Albumin is the major plasma protein target of oxidant stress in uremia[END_REF]. Structural alterations on human serum albumin have been described in renal disease such as uremia and chronic liver disease such as liver cirrhosis [START_REF] Ivanov | Chronic liver and renal diseases differently affect structure of human serum albumin[END_REF]. One of the causes of modification on albumin is oxidative stress [START_REF] Roy | Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin[END_REF] and oxidized albumin has been detected in the blood of patients suffering from diabetes [START_REF] Toyokuni | Serum 4 -hydroxy-2-nonenal-modified albumin is elevated in patients with type 2 diabetes mellitus[END_REF] and kidney disease [START_REF] Himmelfarb | Albumin is the major plasma protein target of oxidant stress in uremia[END_REF]. Oxidative stress has also been demonstrated in conditions such as non alcoholic fatty liver disease (NAFLD) [START_REF] Araya | Increase in long-chain polyunsaturated fatty acid n -6/n -3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease[END_REF] and non alcoholic steatohepatitis (NASH) [START_REF] Koruk | Oxidative stress and enzymatic antioxidant status in patients with nonalcoholic steatohepatitis[END_REF][START_REF] Bahcecioglu | Levels of serum vitamin A, alpha-tocopherol and malondialdehyde in patients with non[END_REF] conditions which can develop from fatty liver. It has been suggested

that albumin, being synthesized in the liver could be exposed to free radicals, resulting in conformational changes which may affect its binding property [START_REF] Ivanov | Chronic liver and renal diseases differently affect structure of human serum albumin[END_REF]. Our initial studies focused on oxidative markers in serum from patients with fatty liver, and the increase in malondialdehyde and protein carbonyl indicate the presence of lipid per oxidation and protein oxidation, suggestive of oxidative stress. But does this result in functional alterations on serum albumin?

Altered cobalt binding to albumin has been reported in myocardial infarction (MI) [START_REF] Bhagavan | Evaluation of human serum albumin cobalt binding assay for the assessment of myocardial ischemia and myocardial infarction[END_REF] myocardial ischemia [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF] and acute coronary syndrome [START_REF] Christenson | Characteristics of a n Albumin Cobalt Binding Test for assessment of acute coronary syndrome patients: a multicenter study[END_REF] and this has been used as a diagnostic test to determine susceptibility to undergo MI [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF]. However, it has also been shown that reactive oxygen species such as super oxide and hydroxyl radicals modify albumin during myocardial disease [START_REF] Roy | Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin[END_REF], suggesting a role for free radicals in inducing change in cobalt binding. Our data indicates that oxidative stress in serum of patients with fatty liver is accompanied by a slight but significant decrease in cobalt binding to albumin. But is oxidative stress and change in cobalt binding linked, or are they two

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12 unrelated observations? ROS generation in vitro has been shown to produce structural changes in human albumin as well as synthetic N-terminus tetra-and octa-peptides from the molecule, with loss of cobalt binding capacity [30]. Our in vitro experiments produced similar results, where exposure to hydrogen peroxide in the presence of copper resulted in a decrease in albumin-cobalt binding. The reversal of this effect with catalase confirms the role of H 2 O 2 in mediating the effect. More specifically, this effect seems to be mediated by the hydroxyl radical, since superoxide generated by the xanthine/xanthine oxidase system had no effect and thiourea and mannitol were able to prevent the decrease in albumin-cobalt binding. Thiourea and mannitol have been used as scavengers for the hydroxyl radical [START_REF] Grady | Hydroxyl radical production during oxidative deposition of iron in ferritin[END_REF] and albumin and other proteins has been shown to be selectively susceptible to hydroxyl radicals in comparison to the superoxide and hydroperoxyl radicals [START_REF] Davies | Protein damage and degradation by oxygen radicals. I. general aspects[END_REF][START_REF] Davies | Protein damage and degradation by oxygen radicals. II. Modification of amino acids[END_REF]. This data is also in accordance with previous studies where simultaneous presence of H 2 O 2 and copper were required to alter cobalt binding of albumin [START_REF] Roy | Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin[END_REF]. The experiments also suggest that copper does not interfere with cobalt binding, probably because they bind at different sites on the protein [START_REF] Roy | Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin[END_REF].

The data so far indicates that oxidative stress in patients with fatty liver results in modification of albumin, such that the protein's binding to cobalt is altered. But what is the mechanism? Binding of transition metals such as copper and nickel is proposed to occur at the N-terminus of the albumin molecule at the amino acid sequence Asp-Ala-His-Lys [START_REF] Sadler | Involvement of a lysine residue in the Nterminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+[END_REF]. Earlier studies examining albumin cobalt binding in the context of cardiovascular disease have used peptides with the N-terminal sequence and shown that it also binds cobalt and loss of the sequence results in decreased cobalt binding [30]. This was suggested as the mechanism for decreased cobalt binding in myocardial infarction

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13 [START_REF] Bar-Or | A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report[END_REF]. However, cobalt may bind to additional sites on albumin [START_REF] Liang | The subsequent effect of interaction between Co(2+) and human serum albumin or bovine serum albumin[END_REF] and a recent study has demonstrated that the principle cobalt binding site on HSA are sites A & B in the interior, with the N-terminal site being of lesser importance [START_REF] Mothes | Evidence that the Principal Co(II)-Binding Site in Human Serum Albumin Is Not at the N-Terminus: Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia[END_REF]. A recent study by Bar-Or et al using plasma from controls & patients with myocardial ischemia indicate that binding to N-terminal may not be the only mechanism for albumin-cobalt binding [START_REF] Bar-Or | The cobalt-albumin binding assay: Insights into its mode of action[END_REF].This indicates that alternate mechanisms for the decrease in cobalt binding exist, involving other ligands binding in the interior of the molecule.

HSA is the primary transporter for the delivery of fatty acids (FA) to tissues, the FA:HSA ratio being between 0.1:1 to 2:1 under physiological conditions and rising to 6:1 or greater in peripheral vasculature during fasting or exercise [START_REF] Spector | Structure and lipid binding properties of serum albumin[END_REF][START_REF] Brodersen | Multiple fatty acid binding to albumin in human blood plasma[END_REF]. The binding of FA to HSA can also increase in pathological conditions such as diabetes, liver disease and cardiovascular disease [START_REF] Cistola | Fatty acid distribution in systems modeling the normal and diabetic human circulation. A 13C nuclear magnetic resonance study[END_REF]. At least seven binding sites for fatty acids have been identified in albumin [START_REF] Simard | Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy[END_REF], saturated fatty acids binding with increasing affinity as their chain length increases [START_REF] Choi | Interactions of very long-chain saturated fatty acids with serum albumin[END_REF]. As mentioned earlier, four different metal binding sites have been identified in albumin [START_REF] Mothes | Evidence that the Principal Co(II)-Binding Site in Human Serum Albumin Is Not at the N-Terminus: Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia[END_REF] and it has been reported that fatty acids and cadmium compete with each other to bind to site A on albumin [START_REF] Stewart | Interdomain zinc site on human albumin[END_REF]. According to Mothes et al cobalt binds to sites A and B on the albumin molecule [START_REF] Mothes | Evidence that the Principal Co(II)-Binding Site in Human Serum Albumin Is Not at the N-Terminus: Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia[END_REF]. But can fatty acids on albumin modulate the protein's cobalt binding activity? Our data shows that this seems to be the case, since removal of fatty acids from HSA results in decreased cobalt binding to the protein.

But what is the relationship between oxidative stress, fatty acids and cobalt binding on albumin in the context of fatty liver disease? Based on the data on oxidative stress and cobalt binding from patients, it seemed that free radicals decrease albumin-

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ACCEPTED MANUSCRIPT 14 cobalt binding, which agrees with the in vitro oxidant exposure experiments. The role of fatty acids in the context is a bit more complex. The in vitro experiments indicate that fatty acid removal compromised cobalt binding; but patients with fatty liver (who had decreased cobalt binding to albumin) showed significant increases in oleic, myristic and lauric acids bound to albumin. The increase in fatty acids on albumin is probably expected, since patients with fatty liver have increased levels of free fatty acids [START_REF] Lewis | Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes[END_REF],

where the rate of FFA uptake is unregulated and therefore directly proportional to plasma FFA concentrations [START_REF] Wahren | Turnover and splanchnic metabolism of free fatty acids and k etones in insulin-dependent diabetics at rest and in response to exercise[END_REF]. Though palmitic acid and stearic acid are present in serum, it has been shown that between bovine & human serum albumin, palmitate binds more tightly to bovine whereas laurate and oleate bind more tightly to human serum albumin [START_REF] Spector | Fatty acid binding to plasma albumin[END_REF], [START_REF] Spector | Binding of long-chain fatty acids to bovine serum albumin[END_REF]. This could be the reason we were unable to detect palmitate on the albumin isolated from controls or patients. The data then suggests that fatty acids have a protective effect in maintaining albumin-cobalt binding, though the in vitro experiments suggest that mechanisms for this protection are independent of oxidative modification.

The protection by increased albumin-fatty acid binding may also explain the relatively small decrease in albumin-cobalt binding in patients with fatty liver. Since conditions such as cardiovascular disease are also associated with increase in serum free fatty acids [START_REF] Most | Free fatty acid metabolism of the human heart at rest[END_REF] and oxidative stress [START_REF] Schnabel | Oxidative stress in cardiovascular disease: successful translation from bench to bedside?[END_REF], it is possible that this mechanism may hold true in cases of myocardial infarction as well.

In conclusion, our studies demonstrate that oxidative stress in serum of patients with fatty liver is associated with a decrease in cobalt binding capacity of albumin. This is accompanied by an increase in fatty acid binding on albumin in these patients, which probably attenuates the effect of free radicals on cobalt binding to the protein.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

  (hydroxymethyl) aminomethane (Tris), purified human serum albumin (HSA), bromo cresol green, dithiothreitol (DTT), 2,4 dinitro phenyl hydrazine (DNPH), 1,1,3,3 tetramethoxy propane, catalase, xanthine and xanthine oxidase were obtained from Sigma Chemical Co., St Louis, MO, U.S.A. Cibachron Blue was from CIBA-GIEGY.

  protein, total cholesterol, triacylglycerol, HbsAg & anti-HCV. The study was approved by the Institutional Research Committee and samples were obtained after informed consent.

  exposure, experiments were repeated with the xanthine/xanthine oxidase system in presence of superoxide dismutase, with & without copper sulfate. Corroborating the results from Figure 3, incubation with

Figure 1 :

 1 Figure 1:-Protein carbonyl (A) and malondialdehyde (B) levels in serum from

Figure 2 :

 2 Figure 2:-Cobalt binding capacity of albumin in serum from controls (£) and

Figure 3 : 2 Figure 4

 324 Figure 3:-Cobalt binding capacity of purified albumin (A) and normal serum (B)

In vitro oxidant exposure:

  For examining the direct effect of oxidative stress on cobalt binding, normal serum (2.5g/dL) as well as purified human serum albumin (2.5g/dL) was treated with either hydrogen peroxide (200µM) and copper sulphate (CuSO 4 ) (200µM), or xanthine (1 mM) and xanthine oxidase (100 mu) for 30 minutes at 37 o C. Following

	ACCEPTED MANUSCRIPT
	centrifugation at 4000 rpm for 10 min. These beads were then washed with Tris HCl
	buffer, following which the bound albumin was eluted with 100ul of Tris HCl
	(0.05M)/NaSCN (0.2M).
	M A N U S C R I P T
	. Briefly, serum samples were incubated with cobalt chloride 0.232 mM (final concentration) and made up to 100 µl with phosphate buffer saline (PBS pH 7.4) in wells of a microtiter plate. This was incubated for 10 min to allow the cobalt to bind to the albumin. At the end of the incubation, the unbound cobalt was measured by reducing cobalt chloride with 1.6 mM DTT (final concentration), adding 200 µl of saline and reading the absorbance at 490nm. Absorbance A C C E P T E D
	readings were also measured in parallel wells, where only cobalt chloride & DTT were
	present, without serum. The amount of bound cobalt was calculated from subtracting the
	value for unbound cobalt from that of the wells which had only cobalt and DTT without
	serum.
	Separation of albumin from serum: For separation of albumin from serum, 100ul
	serum was incubated with 250ul of blue sepharose beads (Cibachron Blue) in Tris Hcl
	buffer (0.05M pH 8) for 30 min, following which the beads were separated by
	6

Acknowledgements

The Wellcome Trust Research Laboratory is supported by the Wellcome Trust, London.

Funding for this study was from the Christian Medical College Fluid Research Fund.

Figure 5:-Fatty acid content on albumin from controls and patients with fatty liver.

Levels of oleic acid (A), myristic acid (B) and lauric acid (C) from albumin extracted from serum of controls (£) and patients with fatty liver (¢). Experiments were carried out as described in the methods section.

The data is expressed as mean ± SEM, n = 5. (*p<0.05).