Caputo Ivana

Maria Barone

Lepretti Vittoria

Martucciello Marilena

Nista Stefania

Troncone Ivan

Auricchio Riccardo

Sblattero Salvatore

Esposito Daniele

Carla

Ivana Caputo

Barone Maria Vittoria

Lepretti Marilena

Martucciello Stefania

Nista Ivan

Troncone Riccardo

Auricchio Salvatore

Sblattero Daniele

Esposito Carla

Prof Carla Esposito
email: cesposito@unisa.it

Celiac anti-tissue transglutaminase antibodies

Keywords: transglutaminase, anti-transglutaminase antibodies, gliadin peptides, celiac disease, endocytosis tTG, tissue transglutaminase, CD, celiac disease, p31-43, peptide 31-43, p57-68, peptide 57-68, PBS, phosphate-buffered saline, PFA, paraformaldehyde, BrdU, bromodeoxyuridine, EGF, epidermal growth factor, ERK, extracellular signal-regulated kinase, FBS, fetal bovine serum, M-β-CD, methyl-β-cyclodextrin, MDC, monodansylcadaverine

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A C C E P T E D

M A N U S C R I P T

Introduction

Celiac disease (CD) is an enteropathy triggered by the ingestion of gliadin and of other related prolamines in genetically predisposed individuals [START_REF] Sollid | Molecular basis of celiac disease[END_REF][START_REF] Sollid | Coelic disease: dissecting a complex inflammatory disorder[END_REF]. Gliadin peptides exert damaging effects through various mechanisms: they are resistant to gastrointestinal enzymes [START_REF] Shan | Structural basis for gluten intolerance in celiac sprue[END_REF], they have amino acid sequences that are specific for human leukocyte antigen (HLA)-DQ2, which is a class II major histocompatibility complex, they also have preferred glutamine residues for tissue transglutaminase (tTG)-mediated deamidation [START_REF] Arentz-Hansen | The intestinal T cell response to gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase[END_REF], and, lastly, they affect intestinal permeability [START_REF] Meresse | Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and lymphomagenesis[END_REF]. Some gliadin peptides are toxic and others are immunogenic for CD patients [START_REF] Meresse | Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and lymphomagenesis[END_REF]. Toxic gliadin peptides exert direct effects on cells. In particular, they can agglutinate undifferentiated K562, can maintain tissue damage in the intestinal mucosa of CD patients, and can activate innate immune mechanisms in patients on a gliadin-free diet [START_REF] Maiuri | Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease[END_REF][START_REF] Gianfrani | Adaptive and innate immune responses in celiac disease[END_REF][START_REF] Maiuri | In vitro activities of A-gliadin synthetic peptides: damaging effect on the atrophic coeliac mucosa and activation of mucosal immune response in the treated coeliac mucosa[END_REF]. On the other hand, immunogenic gliadin peptides, after passing the intestinal epithelial border, trigger a dysregulated adaptive immune response at gut level dominated by T cells of the T helper 1 type. However, the innate and adaptive immune systems may respond synergistically to gliadin peptides [START_REF] Meresse | Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and lymphomagenesis[END_REF][START_REF] Maiuri | Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease[END_REF][START_REF] Gianfrani | Adaptive and innate immune responses in celiac disease[END_REF]. The prototype of peptides effective on innate response is peptide 31-43/49, which has been shown to be toxic for CD patients both in vitro and in vivo [START_REF] Maiuri | In vitro activities of A-gliadin synthetic peptides: damaging effect on the atrophic coeliac mucosa and activation of mucosal immune response in the treated coeliac mucosa[END_REF][START_REF] Ciclitira | In vivo gluten ingestion in coelic disease[END_REF]. stimulates the synthesis and release of the proinflammatory cytokine IL-15 that can promote an adaptive immune response [START_REF] Maiuri | Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease[END_REF] involving CD4+ T cells that recognize various deamidated gliadin peptides [START_REF] Arentz-Hansen | The intestinal T cell response to gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase[END_REF]. Unlike p31-43 which is not immunogenic for T cells, peptide 57-68 (p57-68), which binds to HLA-DQ2/8 molecules, is one of the dominant epitopes recognised by T cells isolated from the intestine of CD patients. [START_REF] Arentz-Hansen | The intestinal T cell response to gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase[END_REF].

Celiac disease is considered an autoimmune disease because of the presence of tTGtargeting autoantibodies in the serum and the intestinal mucosa of patients. tTG is a ubiquitously expressed multifunctional protein [START_REF] Lorand | Transglutaminases: crosslinking enzymes with pleiotropic functions[END_REF] that prevalently catalyzes the formation of isopeptide linkages between the γ-carboxamide group of protein-bound glutamine residue and

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

the ε-amino group of protein-bound lysine residue. Glutamine residues can be deamidated to glutamic acid as a side-reaction in the absence of suitable amines or at low pH. Furthermore, tTG is capable of both binding and hydrolyzing GTP, so that the enzyme can function as a cell signal transducer in association with the the α 1β -adrenoreceptor [START_REF] Im | Transglutaminase II: a new class of GTP-binding protein with new biological functions[END_REF]. Also disulphide isomerase activity [START_REF] Hasegawa | A novel function of tissue-type transglutaminase: protein disulphide isomerase[END_REF] and kinase activity [START_REF] Mishra | Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase[END_REF], have been attributed to tTG.

In CD, anti-tTG autoantibodies are produced in the small-intestinal mucosa and are deposited in the small bowel mucosa below the basement membrane and around capillaries in untreated patients [START_REF] Korponay-Szabo | In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies[END_REF][START_REF] Kaukinen | Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study[END_REF]. Interestingly, even seronegative patients have these mucosal anti-tTG deposits when on a gluten-containing diet [START_REF] Salmi | Endomysial antibody-negative coeliac disease: clinical characteristics and intestinal autoantibody deposits[END_REF]. During gluten consumption, anti-tTG antibodies can be detected also in the serum of patients and slowly disappear from the circulation consequent to a gluten-free diet [START_REF] Stenman | Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits[END_REF]. After a gluten-free diet, serum autoantibodies disappear from the circulation more rapidly than do the small-intestinal mucosal autoantibody deposits [START_REF] Korponay-Szabo | In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies[END_REF][START_REF] Kaukinen | Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study[END_REF].

It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of CD. In fact, they inhibit intestinal epithelial cell differentiation [START_REF] Halttunen | Serum Immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation[END_REF],

increase epithelial permeability and activate monocytes [START_REF] Zanoni | In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes[END_REF], and impair angiogenesis [START_REF] Myrsky | Coeliac disease-specific autoantibodies targeted against transglutaminase 2 disturb angiogenesis[END_REF].

Moreover, we previously demonstrated that anti-tTG antibodies induce intestinal epithelial cell proliferation [START_REF] Barone | Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease[END_REF]. Interestingly, the effect of epithelial cell proliferation evoked by anti-tTG antibodies is similar to the effect evoked by some gliadin peptides [START_REF] Barone | Growth factorlike activity of gliadin, an alimentary protein: implications for coeliac disease[END_REF]. In particular, p31-43, but not p57-68, causes actin alterations and cell proliferation, both of which depend on activation of the epidermal growth factor (EGF)-receptor, in several cell types, and in organ culture of celiac mucosa [START_REF] Barone | Growth factorlike activity of gliadin, an alimentary protein: implications for coeliac disease[END_REF]. These effects of gliadin peptides are regarded as non-Tcell mediated, and most likely play an important role in the very early phases of CD.

However, little is known about the mechanism underlying p31-43 and p57-68 uptake into cells [START_REF] Schumann | Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue[END_REF].

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

The aim of this study was to investigate the relationship between anti-tTG antibodies and gliadin peptides, both of which are present in the small-intestinal mucosa of patients in the early phase of CD.

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

Materials and Methods

Antibodies and peptides -The commercial monoclonal tTG-antibody clone CUB 7402 (200 µg/ml) was from NeoMarkers (Freemont, CA, USA). The recombinant anti-tTG miniantibody (clone 2.8) was derived from a library of CD intestinal lymphocytes [START_REF] Marzari | Molecular dissection of the tissue transglutaminase autoantibody response in celiac in celiac disease[END_REF]. The cloning and purification procedure is reported elsewhere [START_REF] Niro | Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models[END_REF]. into N,N-dimethylated casein, as reported elsewhere [START_REF] Esposito | Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ[END_REF]. Cellular extract from Caco-2 cells (15 µg) was used as enzymatic source and incubated without (basal activity) and with antibodies and amines at 37 °C for 30 min.

Immunoprecipitation of surface tTG from Caco 2 cell membranes -We used the immunoprecipitation strategy (slightly modified) reported by Akimov et al. [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF]. Briefly, Caco-2 cells (about 20x10 6 ) were extensively washed with PBS without calcium and magnesium, and detached with 0.5% EDTA to preserve surface tTG. We biotinylated the cell surface using PBS containing 4 mM Sulfo-NHS-Biotin (Pierce, Milan, Italy) for 30 min at room temperature, and the reaction was quenched with 100 mM glycine in PBS. After washing with PBS, cells were disrupted by osmotic shock in deionized water and the cell membrane fraction was enriched by centrifugation (14,000 g for 90 min). Pellets were resuspended in 0.25 ml of RIPA buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl 2 , 0.1% sodium dodecyl sulphate, 1% triton X 100, 0. Western blot -For the detection of the phosphorylated form of extracellular signal-regulated kinase (P-ERK), cell lysates were prepared as described previously [START_REF] Migliaccio | Tyrosine kinase/p21ras/MAPkinase pathway activation by estradiol-receptor complex in MCF-7 cells[END_REF], as well as electrophoresis and immunoblotting procedures [START_REF] Migliaccio | Tyrosine kinase/p21ras/MAPkinase pathway activation by estradiol-receptor complex in MCF-7 cells[END_REF]. P-ERK was detected using anti-P-ERK mouse monoclonal antibody (clone E4, Santa Cruz). Total ERK was detected using ERK rabbit polyclonal antibody anti-ERK K23 (clone K23, Santa Cruz). The samples resulting from immunoprecipitation experiments were separated by 10% sodium dodecyl sulphate gel electrophoresis and transferred to a membrane. Biotinylated proteins were visualized on the blot with horseradish peroxidase-conjugated streptavidin 1:2000 (Pierce) according to the procedure described elsewhere [START_REF] Zhang | Modulation of the in situ activity of tissue transglutaminase by calcium and GTP[END_REF]. The blot was then reprobed with CUB 7402, as described previously [START_REF] Orrù | Proteomics identification of acyl-acceptor and acyl-donor substrates for transglutaminase in a human intestinal epithelial cell line. Implications for celiac disease[END_REF], to visualize tTG. Immunocomplexes were revealed using the chemiluminescence detection kit (Pierce) according to manufacturer's instructions.

Visualization of peptide uptake by Caco-2 cells -Caco-2 cells were seeded on glass coverslips (5x10 3 /cm 2 ) and three days later were treated for 15 min at 37°C with 100 µg/ml gliadin lissamine-peptides (p31-43 or p57-68). When requested, inhibitors of endocytosis, such as methyl-β-cyclodextrin (M-β-CD) and filipin (Sigma-Aldrich), were added to the medium 15 min before treatment with peptides. To investigate the effect of anti-tTG antibodies on gliadin peptide uptake by cells, 2 µg/ml of clone CUB 7402, or clone 2.8, or non specific IgG, were added to the medium 15 min before treatment with peptides.

Coverslips were then washed with PBS, cells were fixed in 3% PFA for 5 min, and, after washing, coverslips were mounted with Moviol. Confocal images were acquired with a LSM 510 Zeiss microscope. Fluorescence intensity was measured on an area of 5000 µm 2 , randomly selected for every sample.

Endocytosis -Caco-2 cells were seeded on glass coverslips (5x10 3 /cm 2 ) and 3 days later used for endocytosis assay. Endocytosis was carried out with 20 ng/ml EGF-Alexa Fluor 488 (Invitrogen). After washing cells with cold PBS, EGF-receptors were loaded with the ligand at 4°C for 1 hour. Then cells were washed with cold PBS and incubated for 15 min at 37°C with warm medium containing unlabelled EGF. To investigate the effect of anti-tTG antibodies, 2 µg/ml clone 2.8 (or CUB 7402) were added during the incubation at 4°C.

Antibodies were added again during the incubation at 37°C. Finally, coverslips were washed with PBS and cells were fixed with 3% PFA for 10 min. Mounted coverlips were observed with a LSM 510 Zeiss microscope and confocal images were acquired. Fluorescence intensity was measured on an area of 5000 µm 2 , randomly selected for every sample.

Statistics -Statistical analysis was performed where appropriate by using the Student's t test.

Differences were considered to be statistically significant at p < 0.05.

Results

ANTI-tTG ANTIBODIES, AS WELL AS GLIADIN PEPTIDES, INDUCE S-PHASE IN CACO-2 CELLS

We recently demonstrated that anti-tTG antibodies, as well as gliadin-peptide 31-43, induce G 0 → S transition in G 0 -synchronized NIH 3T3 fibroblasts [START_REF] Barone | Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease[END_REF][START_REF] Barone | Growth factorlike activity of gliadin, an alimentary protein: implications for coeliac disease[END_REF]. In the present study, we investigated whether Caco-2 cells, which are human epithelial cells that share several functions with small intestinal enterocytes [START_REF] Hidalgo | Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability[END_REF][START_REF] Goke | Regulation and function of extracellular matrix intestinal epithelial restitution in vitro[END_REF], responded in the same way to tTG antibodies and to gliadin peptides.

First, we verified that the antibodies we used in this study (the commercial CUB 7402 and the recombinant mini-antibody from a CD patient, named "clone 2.8") specifically recognize cell surface tTG on Caco-2 cells. We biotinylated cell surface proteins and specifically immunoprecipitated tTG from a membrane-enriched fraction. With clone 2.8, we used a more bland buffer containing Nonidet P-40 to extract proteins, because clone 2.8 is a conformational antibody and only recognizes a native tTG [START_REF] Sblattero | The analysis of the fine specificity of celiac disease antibodies using tissue transglutaminase fragments[END_REF]. Figure 1A Peptide 57-68 did not induce S-phase entry (40.3±2.1% of BrdU incorporation) when used at a concentration of 100 µg/ml.

ANTI-tTG ANTIBODIES IMPAIR THE CAPABILITY OF P31-43 TO INDUCE CACO-2 CELLS INTO S-PHASE

Because we found that anti-tTG antibodies and p31-43, but not p57-68, drive Caco-2 cells into S-phase, we stimulated intestinal cells with clone 2.8 and p31-43 separately and in combination. As reported in Fig. 3, concentrations of clone 2.8 (2 µg/ml) and p-31-43 (100 µg/ml) induced 47.0±1.0% and 48.7±1.5% BrdU incorporation respectively when used separately and 48.3±2.9% BrdU incorporation when used together (light grey bars). This result suggests that anti-tTG antibodies and p31-43 interacted at some level. Therefore we evaluated the effect of clone 2.8 at a suboptimal concentration (0.2 µg/ml) combined with p31-43 at a concentration that induced the highest BrdU incorporation (100 µg/ml). Under these conditions, BrdU incorporation was reduced to 40.5±0.7%, which suggests that anti-tTG antibodies, at a suboptimal concentration, affected p31-43 stimulation (Fig. 3, white bars). We also tested the effect of clone 2.8 at a concentration that induced the highest BrdU incorporation (2.0 µg/ml) combined with a suboptimal concentration of p31-43 (20 µg/ml). In this case, BrdU incorporation was not affected (46.0±1.0% versus 47.0±0.8% produced by clone 2.8 alone) (Fig. 3, dark grey bars) which indicates that p31-43 did not influence the effects induced by anti-tTG antibodies. We obtained similar results when we used the commercial clone CUB 7402 (not shown) instead of clone 2.8.

RELATIONSHIP BETWEEN tTG'S EXTRACELLULAR TRANSAMIDATING ACTIVITY AND THE EFFECTS EVOKED BY GLIADIN PEPTIDES

We next investigated if the transamidating activity associated with the extracellular membrane surface of tTG could be involved in the modulation of cell cycle progression. To this aim, we carried out an S-phase entry assay with cystamine, a TG irreversible inhibitor, and monodansylcadaverine (MDC), a TG competitive substrate. As shown in Fig. 4A, cystamine did not affect BrdU incorporation. Similarly, MDC did not induce S-phase entry at any of the concentrations tested (Fig. 4B); indeed, BrdU incorporation was slightly reduced at 50 µM. Notably, both amines, like CUB 7402 and clone 2.8, exerted a strong inhibitory effect in an in vitro enzymatic assay (Fig. 4C).

ANTI-tTG ANTIBODIES DERANGE P31-43 (BUT NOT P57-68) INTERNALIZATION BY CACO-2 CELLS

We next evaluated whether anti-tTG antibodies impaired p31-43's capability to drive Caco-2 cells into S phase by interfering with gliadin-peptide uptake by cells. To this aim, we examined the mechanisms of transport in Caco-2 cells using lissamine-labelled p31-43 (p31-43-liss) and p57-68 (p57-68-liss) in the absence and presence of anti-tTG antibodies.

When we pre-treated Caco-2 cells with M-β-CD, a general inhibitor of endocytosis, for 15 min and then added p31-43-liss to the medium and incubated the mix at 37°C for a further 15 min, p31-43-liss entry into Caco-2 cells was dramatically reduced (Fig. 5A). On the contrary, when we pre-treated the cells with filipin, an inhibitor of lipid raft/caveolaemediated endocytosis, p31-43-liss entry into Caco-2 cells was not affected (Fig. 5A). We concluded that p31-43 enters Caco-2 cells by endocytosis and excluded the involvement of lipid raft/caveolae-mediated endocytosis. As shown in Fig. 5B, when we pre-treated cells with M-β-CD, p57-68-liss uptake was slightly reduced, whereas filipin-pretreatment reduced p57-68-liss uptake in a more consistent way (Fig. 5B). Thus, under our conditions, p57-68 was endocytosized by Caco-2 cells, probably through a lipid raft/caveolae-mediated mechanism.

To evaluate whether or not anti-tTG antibodies affect gliadin-peptide uptake into Caco-2 cells, we carried out peptide uptake experiments in the presence of CUB 7402 and clone 2.8. As reported in Fig. 6A, pre-treatment with clone 2.8 (2 µg/ml) resulted in weak p31-43-liss staining, which indicates that the antibody affected the entry of p31-43-liss into the cells. A similar effect was obtained with CUB 7402. Conversely, non specific IgG did not affect p31-43-liss uptake (Fig. 6A). Quantification of the percent of fluorescence intensity

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

Discussion

We investigated the relationship between anti-tTG antibodies and gliadin peptides, both of which are present in the small-intestinal mucosa of patients in the early phase of CD, probably even at luminal level. Our results indicate a hitherto unknown role of celiac anti-tTG antibodies, i.e., they impair the uptake of p31-43, but not of p57-68, by epithelial cells. We previously reported that celiac anti-tTG antibodies and α-gliadin p31-43 induce the G 0 -S transition in murine fibroblasts and enterocytes from celiac patients [START_REF] Barone | Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease[END_REF][START_REF] Barone | Growth factorlike activity of gliadin, an alimentary protein: implications for coeliac disease[END_REF]. We hypothesized that autoantibodies to tTG and gliadin peptides together could produce an additive or synergic proliferative effect in cells of epithelial origin. To test our hypothesis, we first investigated whether the response of Caco-2 cells to tTG-antibodies is similar to that of NIH 3T3 cells and of other cells we previously used [START_REF] Barone | Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease[END_REF]. Here we report that both commercial clone CUB 7402 and a recombinant mini-antibody from a CD patient induce proliferation in Caco-2 cells. Moreover, we found that p31-43 but not p57-68, induce Caco-2 cell proliferation.

Surprisingly, when we stimulated intestinal cells with effective concentrations of p31-43 combined with anti-tTG antibodies, there was no additive effect. On the contrary, suboptimal concentration of antibodies together with effective concentration of p31-43 inhibited BrdU incorporation. In conclusion, our results indicate that anti-tTG antibodies interfered with p31-43 stimulation whereas p31-43 did not affect the response induced by anti-tTG antibodies.

In an attempt to explain these effects, we investigated the relationship between tTGantibodies and gliadin peptide uptake. A critical step in the pathogenesis of CD is the epithelial handling of gliadin peptides. It is known that gliadin peptides enter the cells, interact with the vesicular compartment (M.V. Barone, unpublished) and are transcytosed to the basal part of the cells [START_REF] Schumann | Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue[END_REF][START_REF] Matysiak-Budnik | Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease[END_REF]. However, the mechanism of uptake is not completely understood. Our results on the comparative uptake of two different gliadin fragments (p31-43

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

and p57-68), in the presence of inhibitors of endocytosis, show that p31-43 and p57-68 act differently in relation to entry into epithelial cells. M-β-CD is considered a non-specific inhibitor of endocytosis and can be used to block all endocytic pathways, whereas the antibiotic filipin specifically blocks lipid raft/caveolae-mediated endocytosis [START_REF] Danielsen | Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions[END_REF][START_REF] Orlandi | Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains[END_REF][START_REF] Schnitzer | Filipin-sensitive Caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocutosis, and capillary permeability of select macromolecules[END_REF][START_REF] Awasthi-Kalia | Differential effects of filipin and methyl-beta-cyclodextrin on B cell receptor signalling[END_REF][START_REF] Torgersen | Internalization of cholera toxin by different endocytic mechanisms[END_REF][START_REF] Vidricaire | A clathrin, caveolae, and dynamin-independent endocytic pathway requiring free membrane cholesterol drives HIV-1 internalization and infection in polarized trophoblastic cells[END_REF]. Our confocal images of lissamine-labeled peptides show that both p31-43 and p57-68 enter Caco-2 cells, but while p57-68 uptake was reduced by filipin, which indicates the occurrence of a lipid raft/caveolae-mediated mechanism, p31-43 uptake was not (Fig. 5).

We also show that anti-tTG-antibodies affect only p31-43 uptake. This effect is specific because a non-specific antibody did not alter the transport pathway (Fig. 6).

Interestingly, anti-β1 integrin antibodies had a similar effect on p31-43 uptake, which suggests that the tTG-integrin complex on the cell surface is implicated in the cellular transport of p31-43 but not of p57-68.

Taken together, our results show that cells handle p31-43 and p57-68 differently, which is in line with other differences we identified between the two peptides. In fact, p31-43 and p57-68 are located in different sites during maturation of endocytic vesicles both in Caco-2 cells and in intestinal biopsies from CD patients (M.V. Barone, unpublished), [START_REF] Zimmer | Endocytotic segregation of gliadin peptide 31-49 in enterocytes[END_REF]. Staining with markers of the endocytic pathway showed that p31-43 remains in early endocytic vesicles whereas p57-68-carrying vesicles mature into late endosomes (M.V. Barone, unpublished). Furthermore, studies on the interaction of "toxic" and "immunogenic" gliadin peptides with a membrane-like environment in vitro showed that only p31-43 was able to bind micelles, whereas p56-68 did not appear to interact with them [START_REF] Vilasi | Interaction of 'toxic' and 'immunogenic' A-gliadin peptides with a membrane-mimetic environment[END_REF].

Since gliadin peptides enter the cells by endocytosis and anti-tTG antibodies interfere with p31-43 entry, we evaluated whether anti-tTG antibodies could interfere with endocytosis. Using EGF-Alexa Fluor 488, which is prevalently internalized by cells through a clathrin-mediated endocytosis [START_REF] Alexander | Endocytosis and intracellular sorting of receptor tyrosine kinases[END_REF], we demonstrate that anti-tTG-antibodies interferes with endocytosis. These results provide strong evidence that cell surface tTG is involved in the A C C E P T E D M A N U S C R I P T mechanism of endocytosis. In 1980, Davies et al., reported that tTG was involved in the mechanism of endocytosis [START_REF] Davies | Transglutaminase is essential in receptor-mediated endocytosis of alpha 2macroglobulin and polypeptide hormones[END_REF]. However, at that time, the existence of an extracellular membrane bound form of tTG was unknown.

We previously suggested [START_REF] Barone | Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease[END_REF] that anti-tTG antibodies, by interacting with the extracellular membrane-bound tTG, cause G 0 → S transition in synchronized NIH 3T3 fibroblasts through a phosphorylation pathway. We also hypothesized that tTG catalytic activity was not involved in this process. Although tTG is prevalently an intracellular enzyme mainly localized in the cytosol, relevant amounts of the protein are present also on the cell surface. Externalized tTG is in part removed from the cell surface and in part constitutes the pool of the membrane-bound protein [START_REF] Zemskov | Cell-surface transglutaminase undergoes internalization and lysosomal degradation: an essential role for LRP1[END_REF]. Surface tTG functions often are independent of catalytic activity. However, the issue is highly debated. In fact, owing to the availability of high calcium levels and low GTP concentrations in the extracellular space, tTG should be basically active, although the effects of anchorage to fibronectin and the actions of metalloproteinases have not been fully explored. It has been demonstrated that, being an outside membrane-bound protein, tTG could mediate the interaction of β1 and β2 integrins with fibronectin in a catalytic independent manner [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF]. Through this interaction, tTG could modulate cell-matrix adhesion, spreading, integrin-mediated signaling, and cell migration, proliferation or differentiation [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF][START_REF] Akimov | Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin[END_REF][START_REF] Zemskov | Regulation of platelet-derived growth factor receptor function by integrin-associated cell surface transglutaminase[END_REF]. Remarkably, the cross-linking activity of tTG is not required for inhibition of p31-43 uptake or for its proliferating effect. In fact, neither cystamine nor MDC, which are inhibitors of tTG catalytic activity, affected peptide entry into Caco-2 cells or cell cycle progression. In addition, we provide a direct demonstration that the interaction of tTG antibodies with the extracellular membrane-bound tTG triggers an intracellular response that leads to a rapid increase of ERK phosphorylation (Fig. 2B).

Therefore, the effects we observed probably result from the interaction of antibodies with tTG on the cell surface without the involvement of catalytic activity.

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

CONCLUSIONS

The results we report herein show that anti-tTG antibodies interfere with p31-43 endocytosis, which suggests that cell-surface tTG could play a hitherto unknown role in the regulation of gliadin peptide uptake and, more generally, in endocytosis.

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

7.

  5 mM phenylmethylsulfonyl fluoride, 1 mM ortovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin [Sigma-Aldrich]), or in the same buffer without sodium dodecyl sulphate and triton X 100 but containing 0.1% Nonidet P-40 (Sigma-Aldrich), for 30 min at 4°C. An aliquot of the extracted A C C E P T E D M A N U S C R I P T proteins was tested in an ELISA assay to verify the presence of a biotinylated protein that could be recognized by antibody clone 2.8 (not shown). The remaining surface proteins were immunoprecipitated with CUB 7402 (3.0 µg) or clone 2.8 (3.0 µg). Mouse IgG (3.0 µg) was used in control immunoprecipitation.

  Fig. 2C, p31-43 induced BrdU incorporation in Caco-2 cells in a dose-dependent manner. In fact, BrdU incorporation increased up to about 50% in cells treated with 100 µg/ml p31-43.

Finally, we investigated 13 10

 13 whether cystamine and MDC affected p31-43-induced Sphase entry. Neither 50 µM cystamine together with 100 µg/ml p31-43 (light grey bars) nor A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT µM MDC with 100 µg/ml p31-43 (dark grey bars) significantly inhibited BrdU incorporation versus p31-43 alone (Fig. 4D).

Fig. 1 -

 1 Fig. 1 -(A) Surface biotinylated proteins immunoprecipitated with clone 2.8, CUB 7402 and

Fig. 2 -

 2 Fig. 2-(A) CUB 7402 (black bars) and clone 2.8 (light grey bars) dose-dependently induced

Fig. 3 -Fig. 4 -Fig. 5 -Fig. 6 -

 3456 Fig. 3-Effect of the simultaneous presence of clone 2.8 and p31-43 on BrdU incorporation by Caco-2 cells. Effective concentrations of clone 2.8 and p31-43 (2 µg/ml and 100 µg/ml,

Fig. 7 -

 7 Fig. 7-(A) Endocytic vesicles containing EGF-Alexa Fluor 488 were less abundant in Caco-2 cells after incubation at 37°C for 15 min in the presence of clone 2.8 (2 µg/ml).

Acknowledgements

We are grateful to Jean Ann Gilder for text editing. This work was supported by grant from Fondi di Ateneo per la Ricerca di Base (FARB-ex 60%) and from ELFID.

(mean of 3 experiments) relative to p31-43-liss uptake, in the presence of clone CUB 7402 or clone 2.8 (2 µg/ml), showed that both antibodies reduced fluorescence by about 50%, whereas non specific IgG did not affect fluorescence intensity (Fig. 6C). These effects were less pronounced, but still significant, at lower concentration (0.2 µg/ml and 1.0 µg/ml) of antibodies (Fig. 6C). Pre-treatment with clone 2.8 or CUB 7402 did not affect p57-68-liss entry into Caco-2 cells (Fig. 6B,C). In addition, As expected, cystamine and MDC did not affect either p31-43-liss or p57-68-liss entry into cells (not shown). Interestingly, when we treated cells with antibodies to β1-integrin (10 µg/ml), which is non-covalently complexed with tTG on the cell surface [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF], p31-43 uptake but not p57-68 uptake was reduced (Fig. 6D).

ANTI-tTG ANTIBODIES AND ENDOCYTOSIS

To determine how the tTG antibodies interfered with endocytosis, we studied their effect on EGF internalization by cells evaluated with the labelled-ligand EGF-Alexa Fluor 488, which is prevalently internalized by cells through clathrin-mediated endocytosis [START_REF] Alexander | Endocytosis and intracellular sorting of receptor tyrosine kinases[END_REF].

After incubation at 0°C for 1 hour with and without clone 2.8 (2 µg/ml), cells were treated with EFG-Alexa Fluor 488 at 37°C for 15 min, then fixed. In the presence of clone 2.8, the number of endocytic vesicles containing EGF-Alexa Fluor 488 after 15 min of incubation at 37°C was lower than in cells not exposed to antibodies (Fig. 7A). To quantify fluorescence, we measured its intensity relative to a random area of 5000 µm 2 in each coverslip. As shown in Fig. 7B, there was no significant difference between fluorescence intensity at time zero and at 15 min in the presence of clone 2.8. Similar results were obtained with CUB 7402 (Fig.


S M Rombach

N Dekker

M G Bouwman

G E Linthorst

A H Zwinderman

F A Wijburg

S Kuiper

M A Vd

Bergh Weerman

J E M Groener

B J Poorthuis

A H Zwinder- Man

C E M Hollak

Johannes M F G Aerts
email: j.m.aerts@amc.uva.nl

M A Vd Bergh Weerman

Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Fabry disease (OMIM 301500) is caused by deficient activity of the lysosomal enzyme α-Galactosidase A (E.C. 3.2.1.22) [START_REF] Brady | Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency[END_REF]. The deficiency leads to lysosomal accumulation of glycosphingolipids with a terminal α-galactosyl moiety, in particular globotriaosylceramide (Gb3), also known as ceramide trihexoside (CTH) [START_REF] Sweeley | Fabry's disease : classification as a sphingolipidosis and partial characerization of a novel glycolipid[END_REF]. Primarily affected cells are smooth muscle and endothelial cells, contributing to the predominance of vascular complications in Fabry disease [START_REF] Desnick | The metabolic and molecular bases of inherited disease[END_REF][START_REF] Schiffmann | Fabry disease[END_REF].

Although its inheritance is X-linked, both males and females can be affected by Fabry disease [START_REF] Macdermot | Natural history of Fabry disease in affected males and obligate carrier females[END_REF][START_REF] Scheidt | An atypical variant of Fabry's disease with manifestations confined to the myocardium[END_REF][START_REF] Nakao | An atypical variant of Fabry's disease in men with left ventricular hypertrophy[END_REF][START_REF] Whybra | Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes[END_REF][START_REF] Gupta | The relationship of vascular glycolipid storage to clinical manifestations of Fabry disease: a cross-sectional study of a large cohort of clinically affected heterozygous women[END_REF][START_REF] Deegan | Natural history of Fabry disease in females in the Fabry Outcome Survey[END_REF].

Male patients typically present acroparesthesia as well as angiokeratoma and hypo-or anhydrosis already at a young age. In the third and fourth decade of life, renal, cardiac, cerebral complications, and hearing loss may develop [START_REF] Schiffmann | Fabry disease[END_REF][START_REF] Macdermot | Natural history of Fabry disease in affected males and obligate carrier females[END_REF]. Atypical manifestations have been described for male patients with high residual enzyme activity. In such individuals the disease may be limited to one organ only, such as the heart, or may show a milder course [START_REF] Schiffmann | Fabry disease[END_REF][START_REF] Macdermot | Natural history of Fabry disease in affected males and obligate carrier females[END_REF][START_REF] Scheidt | An atypical variant of Fabry's disease with manifestations confined to the myocardium[END_REF][START_REF] Nakao | An atypical variant of Fabry's disease in men with left ventricular hypertrophy[END_REF]. Intriguingly, despite significant amounts of residual α-Galactosidase A activity, female carriers display a vast spectrum of disease severity, ranging from total absence of clinical disease to severe organ damage [START_REF] Whybra | Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes[END_REF][START_REF] Gupta | The relationship of vascular glycolipid storage to clinical manifestations of Fabry disease: a cross-sectional study of a large cohort of clinically affected heterozygous women[END_REF][START_REF] Deegan | Natural history of Fabry disease in females in the Fabry Outcome Survey[END_REF][START_REF] Wang | Heterozygous Fabry women are not just carriers, but have a significant burden of disease and impaired quality of life[END_REF][START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF]. The observed residual enzyme activity in plasma or blood cells from female carriers varies considerably due to random Xinactivation, ranging from normal to nearly completely absent, and is a poor predictor of the clinical course [START_REF] Linthorst | Enzyme activity for determination of presence of Fabry disease in women results in 40% false-negative results[END_REF]. Also in other aspects, the relationships between residual enzyme activity, Gb3 accumulation and disease manifestations in Fabry patients are still puzzling. For example, it has been documented that in male hemizygotes prominent storage of Gb3 already occurs in utero whilst clear clinical symptoms develop much later in life [START_REF] Popli | Demonstration of Fabry's disease deposits in placenta[END_REF][START_REF] Vedder | Manifestations of Fabry disease in placental tissue[END_REF]. Furthermore, it has become apparent that no strict correlation exists between clinical manifestation of Fabry disease and plasma Gb3 levels [START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF][START_REF] Vedder | Manifestations of Fabry disease in placental tissue[END_REF][START_REF] Bekri | The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature[END_REF]. In hemizygotes with classical manifestation of Fabry disease, the plasma Gb3 level is already abnormally high at very young age prior to prominent clinical symptoms, whereas in heterozygotes, even symptomatic females, Gb3 levels are generally within the normal range. Based on these findings, monitoring of the clinical course in Fabry patients by analysis of their plasma Gb3 is presently considered to be of little value [START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF][START_REF] Young | Is globotriaosylceramide a useful biomarker in Fabry disease?[END_REF][START_REF] Bekri | The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature[END_REF]. In urine of symptomatic Fabry patients, both

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heterozygotes and hemizygotes, Gb3 levels are generally abnormally high [START_REF] Whitfield | Monitoring enzyme replacement therapy in Fabry disease--role of urine globotriaosylceramide[END_REF][START_REF] Blais | Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease[END_REF]. However, again no correlation of this parameter with disease manifestation has been observed [START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF][START_REF] Young | Is globotriaosylceramide a useful biomarker in Fabry disease?[END_REF][START_REF] Bekri | The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature[END_REF].

The enigmatic relationships between residual α-Galactosidase A capacity, Gb3 levels and disease manifestation has prompted us to consider an additional factor in the pathogenesis of Fabry disease.

In analogy to Krabbe disease, the occurrence of a deacylated storage lipid was hypothesized. Indeed, globotriaosylsphingosine (lysoGb3) was found to be dramatically elevated in plasma of all male patients with classical Fabry disease [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. Plasma lysoGb3 was also increased in plasma of symptomatic female patients, although to a lesser extent than in male patients. Comparable findings were made in Fabry mice lacking α-Galactosidase A activity [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF].

In our initial investigation, a relatively small number of Fabry patients were studied, in particular few females [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. In the follow-up investigation presented here, the entire cohort of Dutch patients with classic manifestation of Fabry disease was analyzed with respect to plasma lysoGb3 content. The aim of this study was two-fold. Firstly, the value of plasma lysoGb3 in confirming the diagnosis of Fabry disease was determined. This is relevant for individuals carrying an abnormality in the α-Galactosidase A gene with unknown consequence, or pre-symptomatic female carriers for which demonstration of true Fabry disease is problematic with present laboratory methods (e.g. those with normal plasma or urinary Gb3). The second aim of the investigation was to determine the relationship between plasma lysoGb3 and clinical manifestations of Fabry disease. Our earlier observation that lysoGb3 is capable of promoting proliferation of smooth muscle cells in vitro [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF], suggested that lysoGb3 could contribute to the noted intima media thickening and vascular pathology, and thus may constitute a risk for disease manifestation [START_REF] Macdermot | Natural history of Fabry disease in affected males and obligate carrier females[END_REF][START_REF] Kalliokoski | Structural and functional changes in peripheral vasculature of Fabry patients[END_REF][START_REF] Barbey | Increased carotid intima-media thickness in the absence of atherosclerotic plaques in an adult population with Fabry disease[END_REF][START_REF] Laurent | Arterial remodelling in Fabry disease[END_REF]. The results of our investigation are reported here.

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Methods

Patients.

All 92 Fabry patients, including 69 adult and 23 paediatric patients from 28 families, with classical

Fabry disease seen at the outpatient clinic of the Academic Medical Center from 1999-2008 were included in the study. Classical Fabry disease was defined as a mutation in the α-Galactososidase A gene and the occurrence of typical manifestations of the disease, such as acroparesthesias, angiokeratoma, hypo-or anhydrosis in at least one male family member. Patients with the R112H and P60L mutation did not meet the criteria of classical Fabry disease and were analyzed separately.

Healthy controls, comparable for age were recruited for analysis of plasma lysoGb3. None of the patients received enzyme therapy at the time of plasma lipid analysis. The diagnosis was confirmed by demonstration of deficient leukocyte α-Galactosidase A activity and genotyping [START_REF] Blom | Recombinant enzyme therapy for Fabry disease: absence of editing of human alphagalactosidase A mRNA[END_REF]. All patients gave informed consent. For all patients, a full medical history was obtained as well as a routine physical examination, including height, weight and blood pressure measurements. Body mass index was determined and hypertension was diagnosed in case of a systolic BP ≥ 140 mmHg and/ or a diastolic BP ≥90 mmHg in three consecutive measurements performed on the same arm.

Dyslipidemia was diagnosed according to the Dutch guidelines for dyslipidemia and cardiovascular risk management. Smoking status was scored as current, smoking in the past, or never smoking.

Renal function (eGFR) was estimated using the abbreviated MDRD equation in adults and the Schwartz formula in children [START_REF] Levey | Using standardized serum creatinine values in the modification of diet in renal disease study equation for estimating glomerular filtration rate[END_REF][START_REF] Schwartz | New equations to estimate GFR in children with CKD[END_REF]. Glomerular filtration rate (GFR) measured by simultaneous infusion of iothalamate and hippuran (mGFR) was performed in adult and in most children (n = 50) and Cr 51 EDTA in 2 adult patients. Renal failure was defined as an mGFR < 60 ml/min.

Microalbuminuria was defined as albumin exceeding 30 mg in a 24-hour-urine portion in two consecutive portions taken a least 1 week apart. Proteinuria was diagnosed when total protein count was more than 300 mg in these samples. A standard brain magnetic resonance imaging (MRI) evaluation for presence of (lacunar) infarctions was available from 83 patients. An electrocardiogram

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(ECG) and cardiac ultrasound investigation was available from 87 and 76 patients, respectively. Left ventricular hypertrophy (LVH) was defined as a left ventricular mass (LVmass) of >259 g in males and >166 g in females [START_REF] Levy | Echocardiographic criteria for left ventricular hypertrophy: the Framingham Heart Study[END_REF]. Hearing tests were performed by using conventional audiometry and were available from 79 patients. Hearing loss was defined as a PTA (the average hearing loss in dB at 0.5, 1.0 and 2.0 kHz) > 25 dB of the most affected ear [START_REF] Germain | Patients affected with Fabry disease have an increased incidence of progressive hearing loss and sudden deafness: an investigation of twenty-two hemizygous male patients[END_REF]. The presence of acroparesthesia was obtained by medical history of pain in hands and feet. The presence of angiokeratoma was scored when this was confirmed by physical examination of the skin by a medical professional specialized in Fabry disease. Overall severity of disease was assessed by the MSSI scoring system, a composite score of clinical symptoms [START_REF] Whybra | The Mainz Severity Score Index: a new instrument for quantifying the Anderson-Fabry disease phenotype, and the response of patients to enzyme replacement therapy[END_REF].

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Plasma Gb3 and lysoGb3 measurements.

Quantitation of Gb3 in plasma samples was performed as described previously [START_REF] Groener | HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma[END_REF]. Quantitative measurements of plasma lysoGb3 were performed as described with minor modifications [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. In brief, 100 µl of plasma was extracted with 900 µl of chloroform/methanol 1/2 (vol/vol). The extract was centrifuged for 10 min. at 14,000 × g and the pellet was discarded. To the supernatant, 300 µl chloroform and 450 µl MQ-H 2 O was added, mixed, and centrifuged for 2 min. at 14,000 × g to separate the phases. The upper phase was collected, and the lower chloroform phase was re-extracted with 1.2 ml of methanol/ MQ-H 2 O 1/1 (vol/vol) to quantitatively extract lyso-Gb3. The combined upper phases were dried under nitrogen flow, taken up in 1 ml MQ-H 2 O, and extracted twice with 1 ml of water-saturated 1-butanol. LysoGb3 was recovered from the butanol phase with an overall recovery of >90%. The butanol phase was dried, dissolved in 120 µl of freshly prepared 0.1 M NaOH in methanol and incubated at 37°C for 1 hour. Of this solution, 50 µl was derivatized with 25 µl of ophtaldialdehyde (OPA) reagent (5 mg of OPA, 0.1 ml of ethanol, 5 µl of 2-mercaptoethanol, and 10 ml of 3% boric acid, pH 9.0). The OPA-derivatized lyso-Gb3 was separated by HPLC and identified by fluorescence detection as described previously [START_REF] Groener | HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma[END_REF]. All plasma samples were extracted in duplicate. Quantification was performed by addition of lysoGb3 (Sigma-Aldrich) to normal plasma at concentrations ranging from 0 to 1 mM. The limit of detection of plasma lysoGb3 was 3 nM.

Quantification of lysoGb3 was based on a commercially available standard (Sigma-Aldrich) as described earlier [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. We plan to synthesize a large batch of lysoGb3 ourselves and use this standard in the future, given the observation that commercial lysoGb3 batches show a contamination with N-acetylated lysoGb3.

Electronmicroscopy

After fixation, the material was post fixed in 1% osmiumtetroxide, block-staining with 1% uranyl acetate, one-step dehydration in dimethoxypropane and embedding in epoxyresin LX-112. LM sections were stained with toluidine blue. EM sections were stained with tannic acid, uranyl acetate and lead citrate and examined in an Philips CM10 (FEI).

Photographs were taken with a Morada digital camera, (S.I.S.).

Statistical analysis

Statistical analyses were performed using SPSS 17.0 (IBM, Chicago). Results are expressed as median (range). Differences between groups were assessed by the Mann-Whitney U-test.

Correlations between variables were described by Spearman's rank correlation coefficients. Multiple regression logistic and linear regression was used to determine the association between lysoGb3 and disease parameters adjusted for age as a continuous variable, gender, smoking status, body-mass index and the presence or absence of a history of hypertension, and the presence of other cardiovascular disease including diabetes, hemochromatosis and dyslipidemia [START_REF] Levy | Stratifying the patient at risk from coronary disease: new insights from the Framingham Heart Study[END_REF].

Linear regression was applied to quantify the association between continuous outcome variables and lysoGb3, while logistic regression was used for binary outcome variables. In the former case, B values represent the slope of the linear regression line (y = a+B x) while keeping all other variables constant and reflects the change of the outcome variable when lysoGb3 changes 1 unit (i.e. 10 nM or 100 nM). With logistic regression the odds ratio was determined which reflects the change of the odds of the binary outcome when lysoGb3 changes 1 unit while keeping all other variables constant.

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Results

Diagnostic value of plasma lysoGb3.

The average age of the male (n=37) and female Fabry patients (n=55) did not differ significantly, the median being 33 and 35 years, respectively. As has been previously described, lysoGb3 values were around 15 times higher in males as compared to females [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF], ranging from 51-489 (median 286) in males to 0-99 (median 18) in females (Figure 1). Also, the disease severity was significantly higher in the male Fabry patients than in female patients, reflected in an average higher MSSI score of 21 and 7, respectively.

To determine the usefulness of plasma lysoGb3 measurement for confirmation of the diagnosis of Fabry disease, the concentrations of Gb3 and lysoGb3 were determined in plasma specimens of all 92 subjects as well as healthy controls (Figure 1). In male Fabry patients, a striking increase in plasma Gb3 concentration was observed. Almost all of the male Fabry patients could also be recognized by their abnormally high plasma Gb3 concentration, although in some cases the Gb3 levels were close to

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Age, overall disease severity and plasma lysoGb3 in male and female Fabry patients.

The effect of age on lysoGb3 levels is depicted in Figure 2. In the case of male Fabry patients, no significant correlation was found between lysoGb3 levels and the age of the investigated individuals. This is consistent with a previous observation of a high lysoGb3 level in cord blood of an affected male [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. In female Fabry patients, lysoGb3 levels showed a trend to be higher in older individuals (ρ= 0.25; p=0.06). Similar findings were made when plasma lysoGb3 levels were related to MSSI scores of Fabry patients reflecting overall disease severity (Fig. 3). Male Fabry patients did not show a correlation between lysoGb3 level and MSSI score (ρ= 0.08; p= 0.63). In contrast, female Fabry patients there was a significant, albeit not strict, correlation between plasma lysoGb3 and MSSI score (ρ= 0.53; p<0.0001).

Plasma lysoGb3 and clinical manifestations.

Given the impact of age and gender on disease severity in Fabry patients, multiple regression analysis was performed to assess whether plasma lysoGb3 is an independent contributor to disease manifestations. For this purpose, male and female patients were analyzed separately. Multiple regression logistic and linear regression was used to determine the association between lysoGb3 and disease parameters adjusted for age as a continuous variable, and general cardiovascular risk factors (table 2). For Fabry males parameters are related to an increase in lysoGb3 of 100 nM; for Fabry females we used a smaller increment of 10 nM since the range of lipid abnormality in females is much lower (see Figure 1). Table 2 shows that based upon linear regression analysis plasma lysoGb3

is not associated with the MSSI score in males (1.3 ± 1.0 increase of MSSI score per 100 nM increase of lysoGb3 (p= 0.23)). In contrast, for female Fabry patients a significant association was noted between plasma lysoGb3 and MSSI score (1.6 ± 0.4 increase of MSSI per 10 nM increase of plasma lysoGb3 (p<0.0001)).

Next, specific disease manifestations were analysed. When adjusted for age, plasma lysoGb3 concentration does not appear to correlate with LVmass in males (p=0.53). However, a significant

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influence was noted for the effect on LVmass in female Fabry patients (24.8 ± 5.6 gram increase per 10 nM increase of lysoGb3 (p<0.0001) and 19.5 ± 5.5 (p= 0.001) after adjustment for hypertension) (Table 2). There was no apparent correlation of plasma lysoGb3 concentration with hearing loss, renal failure (both mGFR and eGFR), microalbuminuria, proteinuria, or presence of angiokeratoma (Table 2).

The association of plasma lysoGb3 with the development of white matter lesions and cerebrovascular accidents formed a clear exception. Plasma lysoGb3 concentration contributes independently to the development of these lesions in the brain in male Fabry patients. The odds ratio was 6.1 (95% CI:

1.4-25.9) per 100 nM lysoGb3 increase (p=0.015). In females the odds ratio was 1.2 (95% CI: 0.9-1.9) per 10 nM lysoGb3 increase, which was not significant (p=0.18). Figure 4 illustrates the occurrence of cerebrovascular white matter lesions for male Fabry patients. Most male patients around 35 years develop brain lesions [START_REF] Mitsias | Cerebrovascular complications of Fabry's disease[END_REF]. The ones without such white matter abnormalities all have comparatively low plasma lysoGb3 levels (see Figure 4). In females (not shown), the patient with the highest plasma lysoGb3 had a white matter lesion already at the age of 13.

Exposure to plasma lysoGb3 and clinical manifestations.

Next we examined whether the lifetime exposure to lysoGb3 contributes to the likelihood of presentation of symptoms. Maximum lifetime exposure was estimated by calculating the product of plasma lysoGb3 concentration and the age of an individual. This is very likely an overestimation in the case of female Fabry patients. The calculated cumulative lysoGb3 exposure of individual Fabry patients was compared to the disease severity (see Table 3). For this purpose, patients were stratified as asymptomatic or mildly affected (MSSI 0-19), moderately affected (MSSI 20-39), or severely affected (MSSI >40) [START_REF] Whybra | The Mainz Severity Score Index: a new instrument for quantifying the Anderson-Fabry disease phenotype, and the response of patients to enzyme replacement therapy[END_REF]. Four ranges of exposure values were distinguished: 0-1000, 1000-5000, 5000-10000 and >10000 U (nM lysoGb3 x year). Female patients with a low lysoGb3 exposure of 0-1000 U (n=38), 36 were asymptomatic or mildly affected. The two patients with a low lysoGb3 exposure who had a higher MSSI score suffered from hypertension and obesity. These risk factors

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may have contributed to the higher MSSI score. Of the female patients with a higher lysoGb3 exposure of 1000-5000 (n=17), [START_REF] Wang | Heterozygous Fabry women are not just carriers, but have a significant burden of disease and impaired quality of life[END_REF] The sixth case (37 y, MSSI 6) had LVH and angiokeratoma. In other words, 5 out of the 6 female patients with relatively high lysoGb3 had significant disease symptoms at the time of the lysoGb3 assessment, not reflected in a high MSSI score exceeding 19. Of the male patients (n=37), only a single, 3 year-old individual, had a low lysoGb3 exposure (802 U) together with a low MSSI of 8.

Table 3 shows that the proportion of male patients with severe disease is clearly increasing with higher lysoGb3 exposure. Of the 12 male patients with a modestly higher lysoGb3 exposure of 1000-5000 U, 8 were mildly affected. The remaining 4 patients were moderately affected. Of the 12 patients with a lysoGb3 exposure of 5000-10000, 6 were mildly affected, 5 moderately affected and a single individual severely affected. The latter case was a smoker with hypertension, which may have contributed to his relatively high MSSI. Finally, in the 12 male patients with a very high lysoGb3 exposure exceeding 10000 U, 6 were moderately affected and 5 were severely affected. Only one individual was mildly affected with a MSSI of 17. The percentage of moderately and severely affected patients increased significantly with increasing lysoGb3 exposure in both females (p< 0.001) and males (p=0.008). Finally, the relationship between plasma lysoGb3 exposure and the incidence of white matter lesions was examined more closely. It was found that in male Fabry patients the plasma

R112H and P60L mutation

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A subset of patients did not classify for classical Fabry disease. This concerned 11 patients with the R112H mutation (5 males, 7 females) and 4 patients with the P60L mutation (2 males, 2 females).

Their lysoGb3 ranged from 0-11 nM in females and 0-20 nM in males. Although, the α-Galactosidase A activity in plasma and leukocytes was reduced in most of these individuals, they did not express any of the classical Fabry symptoms. A single 61-year old male from this group developed renal insufficiency at the age of 43. A kidney biopsy performed at that time led to the diagnosis of Fabry disease. Recent reanalysis of that biopsy showed some storage in the epithelial cells, but no storage in the endothelium as is usually detected in Fabry patients (Figure 6). This case was considered an atypical variant of Fabry disease by the pathologist. Furthermore this patient suffered from hypertension that could have attributed to development of renal insufficiency. These observations suggest that in case of a low plasma lysoGb3 values in males a diagnosis of Fabry disease may need reconsideration in individuals with an α-Galactosidase A mutation with unknown consequence.

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Discussion

Our investigation of 92 hemizygotes and heterozygotes for Fabry disease, seen at the Academic Medical Centre prior to therapy, has revealed that measurement of plasma lysoGb3 concentration is useful for confirmation of the diagnosis of Fabry disease. In all hemizygous male and adult heterozygous female Fabry patients the plasma lysoGb3 concentration was found to be abnormally high and clearly distinguishable from normal. In very young female Fabry heterozygotes a lack of plasma lysoGb3 elevation may however be uninformative. For example, we were unable to detect elevated plasma lysoGb3 in a 7-year old Fabry patient. However, at the age of 11 years the plasma lysoGb3 was clearly elevated. The findings for plasma lysoGb3 levels sharply contrast with those for plasma Gb3 concentrations. Confirmation of the diagnosis of Fabry disease by demonstration of elevated plasma Gb3 concentration is only possible in male Fabry patients. Most female Fabry patients show plasma Gb3 levels within the normal range.

Our study further revealed that plasma lysoGb3 is not elevated in a number of individuals carrying an α-Galactosidase A gene which a nucleotide change which is not unequivocally linked to Fabry disease. Examples of this are the R112H substitution and the P60L substitution. Although, the α-Galactosidase A activity in plasma and leukocytes is reduced in these individuals, they do not develop marked elevated plasma lysoGb3. Since their urinary Gb3 is not increased, it seems questionable whether these individuals should be considered as Fabry patients at all. These observations suggest that plasma lysoGb3 measurement may prove to be a useful additional assessment for confirmation of Fabry disease in individuals with an α-Galactosidase A mutation with unknown consequence.

A pathophysiological role of elevated plasma lysoGb3 in Fabry disease may be considered. Barbey and co-workers were the first to report an increased carotid intima-media thickness in the absence of atherosclerotic plaques in adult Fabry patients [START_REF] Barbey | Increased carotid intima-media thickness in the absence of atherosclerotic plaques in an adult population with Fabry disease[END_REF], a finding which was soon confirmed by others [START_REF] Kalliokoski | Structural and functional changes in peripheral vasculature of Fabry patients[END_REF]. Barbey and colleagues proposed that a circulating factor contributes to the noted vessel remodelling in Fabry patients and subsequent vasculopathy [START_REF] Barbey | Increased carotid intima-media thickness in the absence of atherosclerotic plaques in an adult population with Fabry disease[END_REF]. We earlier demonstrated that

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exposure of smooth cells in culture to lysoGb3 concentrations as occurs in male Fabry patients prominently stimulates the proliferation of these cells [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. Our present investigation using multiple logistic and linear regression with adjustments for age and cardiovascular risk factors, revealed some significant relationships between plasma lysoGb3 concentration and clinical manifestations. This is remarkable since relatively low numbers of male (n=37) and female (n=55) Fabry patients were studied. The relationships become more pronounced when data of both genders are combined with appropriate adjustment for gender. For example, the linear correlation between lysoGb3 and MSSI is 1.4 ± 0.5 (p=0.007) per 10 nM increase of lysoGb3 and 24.3 ± 5.3 g increase of LVmass (p <0.0001) per 10 nM increase of lysoGb3 for the patients combined. The logistic correlation between lysoGb3 and white matter lesions shows an odds ratio (95% CI) of 1.2 (1.0-1.3) (p=0.004) for both males and females per 10 nM increase.

We investigated whether lifetime exposure to plasma lysoGb3 influences disease severity. The individual lifetime exposure was crudely estimated by calculating the product of plasma lysoGb3 level and age of the investigated subject. A correlation indeed was noted between plasma lysoGb3 exposure disease classification, using the Mainz severity scoring index (MSSI) [START_REF] Whybra | The Mainz Severity Score Index: a new instrument for quantifying the Anderson-Fabry disease phenotype, and the response of patients to enzyme replacement therapy[END_REF], both for male and female Fabry patients. This finding is striking since white matter abnormalities and micro-albuminuria hardly contribute to the Mainz severity score.

It should be mentioned that age by itself is a major factor that determines the clinical outcome in Fabry patients. The contribution of lysoGb3 on top of age is most apparent with respect to white matter lesions: in patients with a high exposure to lysoGb3, white matter lesions were clearly more frequently present. The inverse was true as well: in the older male patients, only the ones with a relatively low lysoGb3 level had not yet developed white matter lesions. For females, the most important conclusion is that levels are much lower than in males, but when relatively high lysoGb3

values are identified at a young age, this almost invariably relates to the presence of disease signs and/or symptoms.

Unfortunately, no IMT data had been collected prior to therapeutic intervention for most of the Fabry patients included in this study. We therefore are unable to correlate IMT values with plasma lysoGb3

A C C E P T E D M A N U S C R I P T concentration or estimated exposure. Very recently Barbey and colleagues reported a slight elevation in sphingosine-1-phosphate in plasma of Fabry patients [START_REF] Levy | Stratifying the patient at risk from coronary disease: new insights from the Framingham Heart Study[END_REF]. In their report it is suggested that this lipid abnormality may underlie the carotid intima media thickening in Fabry patients. Spingosine-1phosphate is known to be an important regulator of the vessel wall [START_REF] Michel | Vascular effects of sphingolipids[END_REF]. Since lysoGb3 and sphingosine-1-phosphate show structural resemblance it is of interest to investigate the effect of lysoGb3 on sphingosine-1-phosphate signaling in the vessel wall.

It is surprising that our data suggest that plasma lysoGb3 concentration is an independent risk factor for different disease manifestations in males and in females (white matter lesions and left ventricular hypertrophy for females, respectively). It will be of interest to study a larger cohort of FD patients with complete clinical assessment, particularly mildly affected male patients, to establish whether the noted gender-difference truly exists or is caused by the limited number of individuals investigated in our study.

Since almost a decade enzyme replacement therapy, based on chronic intravenous administration of recombinant α-Galactosidase A, has become available for the treatment of Fabry patients [35 -37].

Two enzyme preparations are currently registered. One enzyme is produced by Chinese hamster ovary (CHO) cells with classic recombinant technology (agalasidase β, Fabrazyme) and the other enzyme is produced by cultured human skin fibroblasts with an activated promoter of the α-Gal A gene (agalasidase α, Replagal). Both recombinant enzymes are quite comparable in properties and differ only slightly in glycan composition [START_REF] Blom | Recombinant enzyme therapy for Fabry disease: absence of editing of human alphagalactosidase A mRNA[END_REF]. We earlier reported on responses in plasma Gb3 levels in Fabry patients treated with the two enzyme preparations at several dosing regimens [START_REF] Vedder | Treatment of Fabry disease: outcome of a comparative trial with agalsidase alfa or beta at a dose of 0.2 mg/kg[END_REF].

The impact of enzyme replacement therapy on plasma lysoGb3 concentration is subject of a separate ongoing investigation. Preliminary findings were reported in our previous study [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF], indicating that treatment of Fabry patients with both enzyme preparations gives rise to a reduction, but not complete correction of plasma lysoGb3 [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. Multiple regression analyses revealed that, when adjusted for age and cardiovascular risk factors, the plasma lysoGb3 concentration in male Fabry patients is an independent risk for cerebral white matter lesions and in female Fabry patients for left ventricular Plasma lysoGb3 levels related to age for 37 male (A) and 55 female patients (B) with Fabry disease.

  the upper normal range (Fig 1 upper panel). In the case of female Fabry patients, plasma Gb3 concentration did not allow a clear discrimination from normal subjects: only few individuals showed values slightly above the upper normal range (Fig 1 lower panel). In sharp contrast to the findings with plasma Gb3, almost all female Fabry patients showed plasma lysoGb3 concentrations clearly exceeding normal levels. Only in two heterozygotes aged 5 years and 7 years respectively, lysoGb3 was within the normal range. The latter individual showed a plasma lysoGb3 of 5.3 nM at the age of 11 years, slightly above the upper normal range. These findings indicate that demonstration of high plasma lysoGb3, (> 3 nM), may be used for confirming the diagnosis of Fabry disease. A (near) normal level in very young female individuals however, does however not exclude the status of a carrier of Fabry disease.

  lysoGb3 exposure (Fig 5B) correlates better than age (Fig 5A) with existence of a white matter lesion. In female patients this difference was less pronounced.

ALegendsFigure 1 .

 1 Figure 1. Diagnostic value of lysoGb3 in Fabry disease.

Figure 2 .

 2 Figure 2. Relation of plasma lysoGb3 levels and age.

Figure 3 .

 3 Figure 3. Relation of plasma lysoGb3 with clinical severity of Fabry disease.

Figure 4 .

 4 Figure 4. Correlation between plasma lysoGb3 concentration and incidence of white matter

Figure 5 .

 5 Figure 5. White matter lesion: relation to lysoGb3 exposure and to age.

Figure 6 .

 6 Figure 6.

  lysoGb3 concentration x age; expressed in Units (nM x year) Asymptomatic-mild = MSSI 0-19; moderate = MSSI 20-39; severe = MSSI >40. * 5/6 patients with MSSI 0-19 had significant symptoms; # hypertension, overweight; ##: hypertension, smoker

  were moderately affected (MSSI 20-39), but 6 had a MSSI below 20. These 6 cases were examined in more detail. The first case (35 y, MSSI 10) had proteinuria, but

	no other complications. A recent follow-up MRI examination, at the age of 40, revealed a lacunar
	infarction. The second case (44 y, MSSI 9) had microalbuminuria, modest LVH, white matter lesions
	and hearing loss. The third case (15 y, MSSI 10) had acroparesthesia and several white matter
	lesions. The fourth case (49 y, MSSI 17) showed angiokeratomata, LVH, white matter lesions, and
	microalbuminuria. The fifth case (53 y, MSSI 18) had LVH, white matter lesions and proteinuria.

Table 1 . Baseline characteristics: lysoGb3 and MSSI score

 1

		Males	Females	Males vs
				females
				(p-value)
	Number of patients, N	37	55
	Age in years Median (range)	33 (3.6-64)	35 (2.0-71)	0.9
	MSSI Median (range)	21 (5-59)	7 (0-32)	<0.0001
	LysoGb3 (nM) Median (range) 286 (51-489)	18 (0-99)	<0.0001

Table 2 . Multiple regression analysis of contribution of plasma lysoGb3 to disease manifestations Linear regression

 2

		Males		Females
		B-value	p	B -value	p
		per 100 nM lysoGb3		Per 10 nM lysoGb3
	MSSI	1.3 ± 1.0	0.23	1.6±0.4	<0.0001
	LVmass	-5.1 ± 8.0	0.53	24.8±5.6 *	<0.0001
	Renal function (mGFR)	-7.1 ± 6.8	0.32	0.3±1.6	0.84
	Logistic regression
		Males		Females
		Odds ratio (95% CI)	p	Odds ratio (95% CI)	p
		per 100 nM lysoGb3		Per 10 nM lysoGb3
	MSSI (>20)	0.8 (0.2-2.4)	0.68	1.9 (1.1-3.5)	0.03
	LVH	0.6 (0.2-2.0)	0.41	1.6 (0.9-2.7)	0.11
	WML	6.1 (1.4-25.9)	0.015	1.2 (0.9-1.9)	0.18
	Hearing loss	3.0 (0.9-10.1)	0.07	1.0 (0.4-2.4)	0.99
	Renal failure (mGFR)**	1.5 (0.5-4.5)	0.49	1.3 (0.05-34.6)	0.87
	Microalbuminuria	0.9 (0.5-1.8)	0.80	1.2 (0.9-1.6)	0.32
	Proteinuria	0.9 (0.4-1.7)	0.66	1.0 (0.6-1.5)	0.93
	Angiokeratoma	1.0 (0.6-1.9)	0.92	1.4 (1.0-1.9)	0.07
	Acroparesthesia	2.3 (0.8-7.0)	0.14	1.0 (0.7-1.4)	0.94
	B-value= 19.5±5.5, p= 0.001 when adjusted for hypertension.

Table 3 . LysoGb3 exposure and disease severity.

 3

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Acknowledgements

We would like to acknowledge Dr. P. van Paassen from the Department of Clinical and Experimental Immunology of the Academic Medical Center Maastricht for providing the EM-image of the patient with the R112H mutation. The authors gratefully thank research nurse Els Ormel, for handling of the blood samples.

Disclosure

This is to certify that prof. dr. J.M.F.G. Aerts, prof.