Svenja Riedle

Helena Kiefel

Daniela Gast

Sandra Bondong

Silke Wolterink

Paul Gutwein

Dr Peter Altevogt
email: p.altevogt@dkfz.de

Nuclear translocation and signaling of L1-CAM in human carcinoma cells requires ADAM10 and presenilin/ -secretase activity

Keywords: rafts, nuclear translocation, signaling, presenilin, -secretase

Synopsis

L1-CAM plays an important role in human carcinoma progression. Overexpression promotes tumor cell invasion and motility, growth in nude mice and tumor metastasis. It is feasible that L1-dependent signaling contributes to these effects. However, little is known about the mechanism in tumor cells. We reported before that L1 is cleaved by ADAM metalloprotease and that the cytoplasmic part is essential for L1 function. Here we analyzed closer the role of proteolytic cleavage for L1-mediated nuclear signaling. Using OVMz carcinoma cells and L1transfected cells as model, we find that ADAM10-mediated cleavage of L1 proceeds in lipid rafts and non-raft domains. The cleavage product L1-32 is further processed by presenilin/secretase to release an intracellular domain of 28 kDa (L1-ICD). Overexpression of dominant negative presenilin-1 or use of a specific -secretase inhibitor leads to an accumulation of L1-32. Fluorescent and biochemical analysis reveal nuclear localization of L1-ICD. Moreover, inhibition of ADAM10 and/or -secretase blocks nuclear translocation of L1-ICD and L1dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation similar to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and presenilin/ -secretase is essential for the nuclear signaling of L1 in human carcinoma cell lines.

Introduction

L1 cell adhesion molecule (L1-CAM) is a 200-220 kDa transmembrane glycoprotein of the immunoglobulin (Ig) superfamily. Initially, it was found to be involved in the regulation of cell migration, axon outgrowth and guidance during the development of the nervous system [START_REF] Schachner | Neural recognition molecules and synaptic plasticity[END_REF]. However, more recent studies have shown that L1 also plays a role in the ontogeny of human tumors and its expression is linked to poor prognosis [START_REF] Senner | L1 expressed by glioma cells promotes adhesion but not migration[END_REF][START_REF] Kaifi | L1 is associated with micrometastatic spread and poor outcome in colorectal cancer[END_REF][START_REF] Boo | L1 expression as a marker for poor prognosis, tumor progression, and short survival in patients with colorectal cancer[END_REF][START_REF] Zecchini | The differential role of L1 in ovarian carcinoma and normal ovarian surface epithelium[END_REF]. The mechanism by which L1 contributes to tumor progression is not clearly established. Previous studies have demonstrated that L1 enhances growth of tumor cells in NOD/SCID mice, augments tumor cell motility on extracellular matrix proteins, invasiveness in matrigel assays and promotes formation of liver metastases [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF], [START_REF] Silletti | Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion[END_REF]. Interference with L1 expression by genetic manipulation was found to be growth inhibitory in vitro [START_REF] Primiano | Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements[END_REF]. Similar results were reported for L1 antibodies [START_REF] Primiano | Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements[END_REF][START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. Importantly, a number of studies have demonstrated that L1 can alter gene expression [START_REF] Silletti | Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion[END_REF][START_REF] Primiano | Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements[END_REF][START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. Although ERK activation appears to be required for this process, it is unclear if additional factors are involved.

We demonstrated before that the ectodomain of L1 is cleaved at the plasma membrane by the metalloprotease ADAM10 [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF][START_REF] Gutwein | ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles[END_REF][START_REF] Stoeck | A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44[END_REF]. The involvement of ADAM10 was confirmed in a study using ADAM-deficient fibroblastic cell lines established from knock-out mice [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF]. This investigation suggested that ectodomain cleavage by ADAM10 is followed by intramembrane presenilin/ -secretase-dependent cleavage leading to the generation of a L1 intracellular domain (L1-ICD) [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF]. The process of regulated intramembrane proteolysis (RIP) is an essential step in a variety of signaling pathways [START_REF] Landman | Got RIP? Presenilin-dependent intramembrane proteolysis in growth factor receptor signaling[END_REF]. Nuclear translocation and transcriptional regulation of proteins such as Notch, CD44 and the amyloid precursor protein (APP) were shown to depend on ADAM-mediated cleavage followed by presenilin/secretase activity [START_REF] Landman | Got RIP? Presenilin-dependent intramembrane proteolysis in growth factor receptor signaling[END_REF]. We thus hypothesized that proteolytic processing might contribute to L1-signaling. Lipid rafts are microdomains within the plasma membrane that are enriched in cholesterol and sphingolipids [START_REF] Simons | Lipid rafts and signal transduction[END_REF]. They have gained attention as platforms for the proteolytic processing of several proteins including APP [START_REF] Ikezu | Caveolae, plasma membrane microdomains for alpha-secretase-mediated processing of the amyloid precursor protein[END_REF][START_REF] Riddell | Compartmentalization of beta-secretase (Asp2) into low-buoyant density, noncaveolar lipid rafts[END_REF] and the cellular prion protein [START_REF] Hooper | Roles of proteolysis and lipid rafts in the processing of the amyloid precursor protein and prion protein[END_REF]. Interestingly, it has already been described that members of the L1 family are associated with cholesterolenriched microdomains [START_REF] Olive | The F3 neuronal glycosylphosphatidylinositol-linked molecule is localized to glycolipid-enriched membrane subdomains and interacts with L1 and fyn kinase in cerebellum[END_REF][START_REF] Ren | Palmitoylation of neurofascin at a site in the membrane-spanning domain highly conserved among the L1 family of cell adhesion molecules[END_REF].

Here we have analyzed closer the role of proteolytic processing for L1 signaling. We observed in OVMz cells that ADAM10-mediated cleavage of L1 proceeds in lipid rafts and non-raft domains. In addition, we provide evidence that presenilin/ -secretase activity is involved in further processing of the metalloprotease cleavage fragment. Pharmacological inhibition of ADAMs or -secretase activity blocked nuclear translocation of L1 and abrogated L1-dependent gene regulation. Moreover, specific targeting of ADAM10 and PS1 with siRNA affected transcription of L1-dependent genes. We also demonstrate that overexpression of recombinant L1-ICD mediates gene regulation similar to full-length L1. Our results establish for the first time that proteolytic processing by ADAMs and presenilin/secretase is essential for nuclear signaling of L1 in cancer cell lines.

Experimental

Cells and DNAs

The ovarian carcinoma cell lines OVMz, the stably transfected cell lines HEK293-hL1 and CHO-hL1 were described before [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF][START_REF] Gutwein | Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells[END_REF]. Human pancreatic adenocarcinoma cells PT45-PI were described [START_REF] Sebens Muerkoster | Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells[END_REF]. Plasmids encoding PS1 and the dominant negative mutant (D385N) were obtained from Christian Haass (University of Munich, Germany). A fragment encoding the cytoplasmic part of L1 (L1cyt) from position 1148G to the C-terminus was constructed by PCR and both L1cyt and full-length L1 were inserted into the retroviral vector pBMIres-Puro. The transduction of cell lines with retroviral vectors and selection with puromycin was described before [START_REF] Stoeck | L1 on ovarian carcinoma cells is a binding partner for Neuropilin-1 on mesothelial cells[END_REF]. All cell lines were cultivated in DMEM supplemented with 10% FCS at 37°C, 5% CO 2 and 100% humidity.

Chemicals and antibodies

Antibodies to the ectodomain (L1-11A) or cytoplasmic domain (pcytL1) of human L1 were described [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF][START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. Mab 74 5H7 [START_REF] Schaefer | L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1[END_REF] to the cytoplasmic part of L1 was a kind gift of Dr. Vance Lemmon (University of Miami, Florida). The mAbs to the ectodomains of ADAM10 (MAB1427) and TACE (MAB9301) were from R&D (Wiesbaden, Germany), C-terminal pAbs were from Chemicon International (AB19026 and AB19027, respectively). mAB 11G2 to ADAM10 was from abcam (Cambridge, UK). The antibodies to nucleoporin, BIP/GRP78, rab11, ERK and phospho-ERK were purchased from BD-Transduction (Heidelberg, Germany). The antibody to LAMP-1 was from Santa Cruz (Heidelberg, Germany). The pAb to the C-terminus of presenilin (P7854) was from Sigma (Taufkirchen, Germany). Secondary antibodies were obtained from Dianova (Hamburg, Germany).

The analysis of L1 cleavage was carried out as described previously [START_REF] Gutwein | Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells[END_REF]. Briefly, supernatants of a confluent cell monolayer were collected and subjected to TCA precipitation. Cells were removed from the tissue culture plastic surface with PBS/5 mM EDTA. Cell pellets were lysed in lysis buffer (20 mM Tris/HCl pH 8.0 containing 1%octylglycopyranoside, 150 mM NaCl, 1 mM PMSF), cleared by centrifugation and mixed with two-fold concentrated reducing SDS-sample buffer. SDS-PAGE under reducing conditions and transfer of separated proteins to Immobilon membranes using semi-dry blotting was described before [START_REF] Gutwein | Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells[END_REF]. After blocking with 5% skim milk in TBS, the blots were incubated with the respective primary antibody followed by peroxidase conjugated secondary antibody and ECL detection.

Isolation and analysis of membrane lipid rafts

Cells (approx. 1x10 7 ) were lysed in 10 mM Tris/HCl pH 8.0 containing 1% Triton-X100, 150 mM NaCl, 1 mM PMSF, 1 g/ml aprotinin at 4°C. The lysate was mixed with an equal volume of 85% sucrose (w/v in TBS containing Boehringer complete ) and 0.5 ml was transferred to a centrifuge tube. A step gradient was prepared by overlaying with 3 ml of 35% sucrose in TBS followed by 0.5 ml of 5% sucrose in TBS as described [START_REF] Krauss | Integrin leukocyte function-associated antigen-1mediated cell binding can be activated by clustering of membrane rafts[END_REF]. The gradient was centrifuged for 18 h at 200.000xg in a Beckman SW60 rotor. 0.5 ml fractions were collected from top of the gradient and precipitated by chloroform/methanol as described [START_REF] Krauss | Integrin leukocyte function-associated antigen-1mediated cell binding can be activated by clustering of membrane rafts[END_REF]. Samples were dissolved in SDS-sample buffer. SDS-PAGE, Western blotting and ECL was done as described [START_REF] Gutwein | Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells[END_REF].

siRNA transfection siRNAs targeting L1, ADAM10 and TACE were described [START_REF] Stoeck | A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44[END_REF][START_REF] Stoeck | L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells[END_REF]. siRNA specific for PS1 (AAGGUCCACUUCGUAUGCUGGTT) was purchased from Eurofins MWG (Eurofins MWG GmbH, Ebersberg, Germany). OVMz cells were transfected with Oligofectamine (Invitrogen) according to manufacturer's protocol.

Quantitative real-time PCR

RNA was isolated using the Qiagen RNAeasy mini kit (Qiagen Hilden, Germany). For qRT-PCR the cDNA was purified on Microspin G-50 columns (Amersham Biosciences, Freiburg, Germany) and quantitated by NanoDrop spectrophotometer (ND-1000, Kisker-Biotechnology, Steinfurt, Germany). Primers for qRT-PCR were designed with the DNA Star Program and were produced by MWG (Ebersberg, Germany). -actin was used as an internal standard. The PCR reaction was performed with the SYBRgreen mastermix (Applied Biosystems, Darmstadt, Germany). The sequences of primers used are available on request.

Flow cytometry and immunofluorescence microscopy

The staining of cells with mAbs and PE-conjugated secondary antibodies has been described [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF]. Cells were analyzed with a FACScan using Cellquest software (Becton & Dickinson, Heidelberg, Germany). For confocal microscopy cells were fixed with 3% PFA-PBS for 20 min, quenched for 10 min in 50 mM NH 4 Cl, permeabilized for 5 min with methanol (-20°C) and stained with pcytL1 and Alexa488-conjugated anti rabbit IgG. Cell preparations were mounted in Mowiol and images were acquired using a confocal laser imaging system (LSM 510; Carl-Zeiss MicroImaging, Inc.). Quantification was performed using ImageJ software. Nuclei were bordered in phase contrast and the pixel of nuclear staining was determined.

Nucleus purification

Nuclei purification was done as described [START_REF] Schmidt-Zachmann | Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components[END_REF]. Nuclear extracts were prepared by incubation of the purified nuclei in high salt buffer (0.42 M NaCl, 20 mM Hepes pH 7.9, 25 % Glycerol) for 30 min at 4°C followed by centrifugation. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Statistical analysis

Results

Cleavage of L1 in lipid rafts and non-raft domains

L1 is expressed as full-length molecule (L1-220) but can be proteolytically cleaved at different sites as outlined in Fig 1A . Cleavage in the third FNIII domain generates a fragment of 85 kDa (L1-85). In addition, a 32 kDa fragment results from membrane proximal cleavage mediated by ADAM10 (L1-32). We initially investigated whether L1-220 was associated with lipid rafts using TX-100 solubilization and sucrose density centrifugation. In OVMz cells a significant portion of full-length L1-220 (between 20-30%, n 10) was recovered in the lowdensity detergent-resistant membrane (DRM) fraction, identified by the marker fyn (Fig. 1B). The L1-32 cleavage fragment was detected in both lipid raft (fraction #2) and non-raft fractions floating above the loading zone (fraction #7). A similar distribution of L1-220 and L1-32 was found in other cell lines such as the breast carcinoma cell line AR [START_REF] Gutwein | Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells[END_REF], stably transduced CHO-hL1, HEK293-hL1 and PT45-PI-hL1 cells (not shown).

Cleavage of L1 by ADAM10 in lipid rafts and non-rafts

The above results indicated that L1 distributes between lipid raft and non-raft domains and that membrane proximal cleavage is apparent in both. We next analyzed the distribution of ADAM10, the protease responsible for constitutive shedding of L1. A subpopulation of mature ADAM10 (66 kDa) was associated with lipid rafts and co-localized with L1-32 (Fig. 1B). In addition, ADAM10 was detected in the bottom fraction of the gradient. To test whether ADAM10-mediated processing is restricted to lipid rafts we targeted ADAM10 with specific siRNA. As control, cells were treated with siRNA to ADAM17 that catalyzes the induced shedding of L1 [START_REF] Stoeck | A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44[END_REF]. Efficient knock-down was confirmed by flow cytometry (Fig. 1C) and Western blot analysis (data not shown). Down regulation of ADAM10 but not ADAM17 reduced the level of L1-32 in both lipid raft and non-raft fractions (Fig. 1D). These results suggested that constitutive shedding of L1 in OVMz cells is independent of lipid raft localization.

L1-32 cofractionates with late endosomal marker LAMP-1

We noticed that fraction #7 of the raft gradient contained L1-32 whereas L1-220 was hardly detectable (Fig. 1B). To further define the composition of fraction #7, we used an established fractionation protocol to separate early and late endosomes and heavy membranes (Fig. 2A) [START_REF] Gorvel | rab5 controls early endosome fusion in vitro[END_REF]. L1-32 was enriched at the 8/25% interface where it cofractionated with the late endosomal marker LAMP-1 (Fig. 2B). The marker protein EEA-1 was not detected, suggesting that fraction #7 was devoid of early endosomes. In contrast, rab11, a marker for recycling endosomes, was detected in the 25%/35% interface. Cofractionation of LAMP-1 and L1-32 implicated that late endosomes/lysosomes are candidate sites for proteolysis of L1.

L1-32 is a substrate for presenilin/ -secretase activity

Earlier work in fibroblastic mouse cell lines had shown that L1-32 undergoes further processing by presenilin/ -secretase [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF]. Active -secretase complex is associated with lipid rafts [START_REF] Wada | Gamma-secretase activity is present in rafts but is not cholesterol-dependent[END_REF]. The PS1 holoprotein (48 kDa) is endoproteolysed to generate N-terminal (25-25 kDa) and C-terminal (18 kDa) fragments. Both fragments are found in high molecular weight complexes with other integral membrane proteins which together constitute the activesecretase. When we analyzed the distribution of presenilin-1 (PS1) in sucrose gradient fractions, the 48 kDa holoprotein was detected in lipid rafts, whereas the 18 kDa C-terminal fragment was mainly found in the bottom fractions (Fig. 3A). We used the specific presenilin inhibitor IX (DAPT) to investigate cleavage of L1 in OVMz cells. The treatment lead to an increase in L1-32 (Fig. 3B). This was not due to enhanced ADAM10 cleavage but rather the effect of blocking L1-32 processing as the amount of soluble L1 in the medium was not increased (Fig. 3C). In agreement with this notion, the accumulation of L1-32 by DAPT was also apparent when metalloprotease cleavage was blocked by the inhibitor TAPI-0 ruling out the possibility that DAPT activated itself metalloproteinase cleavage (data not shown). Overexpression of PS1 lead to a significant reduction of L1-32 in both lipid raft and non-raft domains. In contrast, dominant negative PS1 (D385N) caused the accumulation of L1-32 (Suppl. Fig 1) in support of a role of presenilin-1 in L1-processing. It was reported before that presenilin/ -secretase cleavage of L1-32 released a smaller Cterminal fragment of 28 kDa in mouse fibroblasts [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF]. In lysates of OVMz or HEK293-hL1 cells L1-28 was difficult to detect most likely due to rapid degradation and/or low abundance. Previous studies have indicated that the intracellular domains (ICDs) of -secretase cleaved proteins are rapidly degraded by cytosolic proteases [START_REF] De Strooper | A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain[END_REF][START_REF] Edbauer | Insulindegrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD)[END_REF]. To directly proof that L1-32 is a substrate for -secretase, we analyzed the processing of L1-32 in microsomal membranes to exclude the degradation by cytosolic proteases. CHO-hL1 cells were cultivated for 16 h in the presence of DAPT or DMSO and membranes were isolated and assayed for in vitro -secretase activity [START_REF] Marambaud | A CBP binding transcriptional repressor produced by the PS1/epsiloncleavage of N-cadherin is inhibited by PS1 FAD mutations[END_REF]. Isolated membranes were incubated for 2 h at 37°C and the membranous and soluble fractions were separated by ultracentrifugation. We identified L1-28 in the supernatant fraction (Fig. 3D, lane 2) of membranes isolated from DMSO treated cells. In contrast, no such fragment was detected in membranes prepared from cells incubated with the presenilin inhibitor. Instead, L1-32 was found in the membrane pellet (Fig. 3D, lane 3). Similar observations were made in OVMz cells (data not shown). These findings suggested that L1-32 is further cleaved by -secretase resulting in the release of L1-28, representing the intracellular domain of L1 (L1-ICD) without membrane portion.

Nuclear accumulation of L1-ICD

Consecutive cleavage by both enzymes is a hallmark of Notch, APP and CD44 signaling that is followed by translocation of the intracellular domain to the nucleus [START_REF] Landman | Got RIP? Presenilin-dependent intramembrane proteolysis in growth factor receptor signaling[END_REF]. As L1 affects gene expression, we investigated the nuclear localisation of L1-ICD. Nuclei were purified using a well established protocol [START_REF] Schmidt-Zachmann | Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components[END_REF]. The purity of the nuclear fractions was examined by Western blot using marker proteins. A representative purification is shown in Fig. 4A. We observed that isolated nuclei were not contaminated by cytosolic proteins (marker moesin) or ER specific proteins (BiP/GRP78 marker). Nucleoporin (nuclear marker) was exclusively present in the nuclear fraction. Using pcytL1 antibody to the cytoplasmic portion of L1, we detected L1-ICD in the nuclear fraction of OVMz (Fig. 4B). When isolated nuclei were extracted with high salt and separated into soluble and insoluble fractions by centrifugation, L1-ICD was found exclusively in the soluble fraction. The L1-ICD fragment was also detected in HEK293-hL1 but was absent in L1-negative HEK293 cells (Fig. 4C).

Nuclear translocation of L1 is blocked by inhibitors of metalloproteases and presenilin/secretase

We performed confocal microscopy to verify the nuclear localization of L1. Using pcytL1 to the cytoplasmic portion of L1, staining was detected in the cytoplasm and the nucleus of CHO-hL1 and OVMz cells that was absent in CHO control cells (Fig. 4D). Next, we studied whether the inhibition of metalloprotease and/or presenilin activity could block L1 nuclear translocation. Treatment with the single compounds or in combination caused an decrease of L1 staining in the nucleus (Fig. 5A). Quantification by image analysis revealed a timedependent decrease of L1 nuclear staining that was detectable after 48 h of treatment and was even more pronounced after 72 h (Fig. 5B, C andD).

Metalloprotease and presenilin cleavage are essential for L1-mediated gene regulation

We investigated whether inhibition of L1-processing affects L1-dependent transcriptional regulation. We have recently identified a set of transcripts in HEK293 cells including CRABPII, beta integrin and CathepsinB that are regulated by expression of L1 [START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. HEK293 and HEK293-hL1 cells were grown for 96 h in the presence of DAPT, TAPI-0 or both inhibitors. QRT-PCR analysis showed that L1-regulated transcription of CathepsinB and CRABPII was blocked by TAPI-0 or DAPT or both (Fig. 6A).

As ERK activation appears to be required for L1-mediated transcriptional regulation, we analyzed the phosphorylation of ERK in treated cells. Importantly, both compounds did not affect ERK activation in HEK293 or HEK293-hL1 cells (Fig. 6B). These results clearly showed that the changes in L1-mediated gene regulation were independent of ERK.

Cleavage of L1 is essential for L1-mediated gene regulation in carcinoma cells

To extend the above findings on ovarian carcinoma cells, we transiently down regulated L1 in OVMz cells (Fig. 7A) and screened for genes that were differentially regulated compared to mock transfected cells. DNA-array analysis identified 282 genes out of 46713 that were up-or downregulated (S. Wolterink, unpublished results). L1-dependent expression of a set of genes was confirmed by qRT-PCR. Fig. 7B shows altered expression levels of transcripts for MDK and beta3 integrin after knock-down of L1 by specific siRNA. To corroborate that consecutive cleavage of L1 is essential for L1-mediated gene regulation, OVMz cells were transfected with siRNAs targeting ADAM10 or PS1. Efficient downregulation of ADAM10 and PS1 was confirmed by western blot (Fig. 7C). Targeting of PS1 caused an accumulation of L1-32 (data not shown) in agreement to what was observed after overexpression of PS1DN (see suppl. Fig. 1) or treatment with DAPT (see Fig. 3B). Downregulation of ADAM10 or PS1 affected transcription of MDK and beta3 integrin similar to downregulation of L1 (Fig. 7D).

Recombinant L1-ICD mediates transcriptional regulation

Based on these findings we hypothesized that an artifical L1-ICD might be able to promote L1-dependent transcriptional regulation. To test this, we constructed a cytoplasmic fragment of L1 lacking the ectodomain and transmembrane region (L1cyt). Cells were stably transduced with L1cyt and full-length L1. Intracellular staining using mAb 745H7 to the cytoplasmic portion of L1 followed by flow cytometry showed that both constructs were expressed at comparable levels (Fig. 8A). Western Blot analysis demonstrated that the L1cyt fragment had a size of appr. 30 kDa and migrated slightly smaller than the L1-32 fragment generated by proteolytic cleavage of full-length L1 (Fig. 8B). Biochemically, L1cyt was detectable in purified nuclei and was very similar in size to the L1-ICD fragment released after -secretase mediated cleavage of L1-32 (Fig. 8C). Importantly, L1cyt affected gene expression of CRABPII and beta3 integrin similar to full-length L1, as shown by qRT-PCR. CathepsinB gene expression was not altered in PT45-PI cells by both L1cyt and full-length L1 (Fig. 8D).

Discussion

The cell adhesion molecule L1 is a type 1 transmembrane protein that is expressed in human carcinomas and melanomas and has been linked to poor prognosis [START_REF] Thies | Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma[END_REF][START_REF] Fogel | L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas[END_REF]. L1 undergoes regulated proteolysis at the cell surface and in released exosomes, a process that involves the metalloprotease ADAM10 [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF][START_REF] Gutwein | ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles[END_REF][START_REF] Stoeck | A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44[END_REF]. Both, L1 and ADAM10, were detected at the invasive front of human colon cancer tissue [START_REF] Gavert | L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers[END_REF] and transfection of ADAM10 confers metastatic capabilities in colon cancer cells expressing L1 [START_REF] Gavert | Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis[END_REF]. In addition, ADAM10 is expressed in ovarian and uterine carcinomas in which soluble L1 is detectable in the sera [START_REF] Fogel | L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas[END_REF].

Little is known about the fate of L1-32 that remains after membrane proximal cleavage. Numerous studies have shown that residual transmembrane remnants become susceptible to RIP. Many substrates for presenilin dependent -secretase have already been identified such as APP [START_REF] Esler | A portrait of Alzheimer secretases--new features and familiar faces[END_REF], Notch [START_REF] De Strooper | A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain[END_REF], ErbB4 [START_REF] Ni | gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase[END_REF], [START_REF] Lee | Presenilin-dependent gamma-secretase-like intramembrane cleavage of ErbB4[END_REF], E-cadherin [START_REF] Marambaud | A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions[END_REF] and CD44 [START_REF] Okamoto | Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway[END_REF]. They all undergo ectodomain shedding which evidently is a prerequisite for subsequent -secretase cleavage. Likewise, recent studies in mouse fibroblastic cell lines demonstrated that L1 is further processed by the presenilin/ -secretase complex after initial cleavage by ADAM10 [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF].

Here we provide evidence that regulated intramembrane proteolysis of L1 is also apparent in ovarian carcinoma cells. The reduction of presenilin/ -secretase activity by the specific inhibitor DAPT or the overexpression of dominant negative PS1 (D385N) lead to the accumulation of L1-32 without concomitant increase of the L1-ectodomain. Moreover, we observed in an in vitro assay that the conversion of the initial ADAM10 cleavage fragment L1-32 into a soluble form of approx. 28 kDa is sensitive to the -secretase inhibitor DAPT. In contrast to the study of [START_REF] Maretzky | L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth[END_REF] we were not able to detect L1-28 in cell lysates, even when cells were grown in the presence of proteasome inhibitors (data not shown). This might be due to low abundance and/or rapid degradation of the L1 intracellular domain in carcinoma cells. However we identified an L1 cleavage product after enrichment of purified nuclei. The size of this fragment was similar to the soluble L1-form released in the in vitro assay described.

We further analyzed the role of proteolytic processing for L1-dependent signaling. Confocal microscopy confirmed that L1-ICD can localize to the nuclei of CHO-hL1 cells. Nuclear translocation was clearly diminished in cells treated with TAPI-0 and DAPT, inhibitors of metalloproteases and -secretase, respectively. These findings suggested that L1 enters the nucleus after initial ADAM10 cleavage followed by RIP. We assumed that L1-ICD in the nucleus might participate in transcriptional regulation in analogy to what has already been described for Notch [START_REF] Kopan | Notch: a membrane-bound transcription factor[END_REF]. To address this question, we investigated the impact of blocking proteolytic processing on L1-dependent gene regulation. We have recently identified a number of genes that are regulated by L1 in HEK293 cells, such as CathepsinB, beta3 integrin, CRABPII or HOXA9 [START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. As demonstrated here for CathepsinB and CRABPII, L1mediated gene regulation was blocked in the presence of specific ADAM and presenilin/secretase inhibitors. Moreover, specific targeting of ADAM10 or PS1 with siRNA affected the transcription of genes, e.g. MDK and beta3 integrin, similar to knock-down of L1 in ovarian carcinoma cells. Taken together, these results highlight the relevance of consecutive processing for the downstream signaling of L1.

To corroborate the role of the L1-ICD in transcriptional regulation, we expressed an artificial L1-ICD lacking the ectodomain and transmembrane region (L1cyt). Indeed, we found that L1cyt localized to the nucleus and mediated transcriptional regulation similar to full-length L1. Additional analysis has shown that L1cyt does not support cell proliferation in vitro and tumor growth in mice as full-length L1 (H.Kiefel unpublished results). These results are in agreement with a recent report [START_REF] Gavert | Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis[END_REF]. Nevertheless, our findings underline the role of the cytoplasmic part of L1 for signaling and establish a link between proteolysis, nuclear translocation and L1-mediated transcriptional regulation.

It is currently not clear how L1 signal transduction by RIP relates to the L1-dependent ERK signaling pathway previously characterized [START_REF] Silletti | Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion[END_REF][START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF][START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. ERK1/2 are serine-threonine kinases that phosphorylate many cellular proteins i.e. transcription factors, cytoskeletal proteins, membrane proteins and other kinases [START_REF] Schaeffer | Mitogen-activated protein kinases: specific messages from ubiquitous messengers[END_REF][START_REF] Colucci-D'amato | Chronic activation of ERK and neurodegenerative diseases[END_REF]. Activated ERK translocates from the cytoplasm to the nucleus where it regulates gene transcription [START_REF] Colucci-D'amato | Chronic activation of ERK and neurodegenerative diseases[END_REF]. Of note, in previous work we have characterized a cytoplasmic L1 mutant (hL1mutTS) devoid of the ERK phophorylation site (Ser1248) that does not support L1-dependent gene regulation and ERK activation [START_REF] Gast | The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies[END_REF]. We observed recently that L1mutTS-ICD does not translocate to the nucleus, although it is processed by ADAM10 [START_REF] Gast | The RGD integrin binding site in human L1-CAM is important for nuclear signaling Exp[END_REF]. In the present paper we found that DAPT or TAPI treatment of L1-expressing HEK293 cells did not affect ERK phosphoryalation (Fig. 6B). Similar observations were made when L1 was specifically depleted by siRNA [START_REF] Stoeck | L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells[END_REF]. Although these results might suggest a less important role of ERK activation, it is still possible that L1 and ERK have to co-operate for L1-processing and/or transcriptional regulation. Further studies have to uncover the contribution of each mechanism to the outcome of L1 signaling.

As lateral segregation of membrane proteins from their sheddases by cholesterol-rich microdomains seems to be a mechanism to control proteolysis [START_REF] Huovila | Shedding light on ADAM metalloproteinases[END_REF], we investigated the importance of L1 partition into lipid rafts for its ectodomain shedding. Using sucrose density gradient centrifugation we showed that L1 distributes between lipid raft und non-raft domains. Moreover, we detected the proteolytic fragment L1-32 in both lipid raft and non-raft fractions. Knock-down of ADAM10 diminished the cleavage fragment in both membrane domains whereas down regulation of TACE had no effect. These data indicated that L1 is cleaved by ADAM10 independently of its distribution into lipid rafts. A similar finding was described for MT1-MMP, a membrane anchored protease that is proteolytically released from the cell surface. The latter process is independent from lipid rafts as both transmembrane and glycosyl-phosphatidylinositol (GPI)-anchored MT1-MMP are shed [START_REF] Toth | MT1-MMP shedding involves an ADAM and is independent of its localization in lipid rafts[END_REF]. Although partition of L1 into lipid rafts seems not to be crucial for its ectodomain shedding, disruption of lipid raft integrity by the cholesterol-binding drug nystatin completely abrogated L1 processing in these microdomains (S.Riedle, unpublished).

L1-32 was also detected in non-raft domains floating above the loading zone (Fig. 1B). Refined subcellular separation demonstrated that L1-32 cofractionated with LAMP-1, a marker for late endosomes/lysosomes. In contrast, full-length L1 was hardly detectable in this fraction. L1-32 may be delivered to late endosomes/lysosomes selectively. It is also conceivable that full-length L1 is completely cleaved within these compartments. Growing evidence suggests a role for endosomes in proteolysis of membrane proteins [START_REF] Pillay | Endolysosomal proteolysis and its regulation[END_REF]. Further analysis has to define the subcellular localization of L1-cleavage. Nevertheless, identification of L1-32 in late endosomes/lysosomes suggests that these endocytic compartments are likely candidate sites for the proteolytic processing of L1. Interestingly, active -secretase complex is also present in lipid rafts although it seems not to be cholesterol dependent [START_REF] Wada | Gamma-secretase activity is present in rafts but is not cholesterol-dependent[END_REF]. We therefore investigated if the presenilin/ -secretase dependent processing of L1 is associated with lipid rafts. Using sucrose density gradient centrifugation we did not detect considerable amounts of PS1-CTF in the lipid raft fraction. This might be caused by the detergent used for the preparation, since the choice of detergent is critical for the detection of PS1 in lipid rafts. Mature components of the -secretase complex are soluble in Triton X-100 but remain insoluble in Lubrol-WX [START_REF] Vetrivel | Spatial segregation of gamma-secretase and substrates in distinct membrane domains[END_REF]. Nevertheless, we observed an increase of L1-32 in the lipid raft fraction after DAPT treatment. Importantly, this accumulation was not restricted to lipid rafts but was also apparent in non-raft domains. Overexpression of dominant negative PS1 (D385N) confirmed these results. A similar finding was noticed in embryonic mouse brains where the majority of PS1, nicastrin and APP-CTF remains soluble in Lubrol-WX [START_REF] Vetrivel | Spatial segregation of gamma-secretase and substrates in distinct membrane domains[END_REF]. Therefore our data support the notion that presenilin/secretase cleavage is not restricted to proteins that are localized in lipid rafts.

The biological meaning of the lateral segregation of L1 into lipid rafts and non-raft domains is presently unclear but has been reported in neuronal cells for L1 and the related molecule neurofascin [START_REF] Nakai | Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner[END_REF][START_REF] Ren | Palmitoylation of neurofascin at a site in the membrane-spanning domain highly conserved among the L1 family of cell adhesion molecules[END_REF]. Our preliminary results suggest that raft resident L1 associates with caveolin-1 (S. Riedle, unpublished results). It should be noted that endocytosis of L1 via the clathrin-mediated pathway has been well characterized [START_REF] Kamiguchi | The neural cell adhesion molecule L1 interacts with the AP-2 adaptor and is endocytosed via the clathrin-mediated pathway[END_REF]. It is therefore tempting to speculate that distinct endocytic pathways are involved in the internalization of L1. Such different routes regulate the signaling and turnover of TGF -receptor [START_REF] Di Guglielmo | Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover[END_REF] and bone morphogenic protein (BMP) receptor [START_REF] Hartung | Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling[END_REF]. Further work has to clarify whether the segregation of L1 into lipid rafts and non-raft regions is required for its internalization via caveolae/raft vs. clathrin dependent pathways. domain. RIP: regulated intramembrane proteolysis.

Legends to Figures Figure 1: L1-cleavage in OVMz cells is mediated by ADAM10

(A) Schematic illustration of L1 cleavage fragments (PPC: proprotein convertase) (B) OVMz cells were lysed in 1% Triton X-100. The sample was mixed with an equal volume of 85% sucrose, placed on the bottom of a centrifuge tube and overlaid with a stepwise sucrose gradient as described in material and methods. After ultracentrifugation fractions were harvested from the top of the gradient and analyzed by SDS-PAGE and Western blotting. Fractions were probed with pcytL1 to the cytoplasmic portion of L1 followed by peroxidaseconjugated secondary antibodies and ECL detection. The nomenclature of L1-cleavage fragments is according to a previous publication [START_REF] Mechtersheimer | Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins[END_REF]. Gradient fractions were reprobed with antibodies to fyn and ADAM10. The mature form (66 kDa) and the proform (97 kDa) are indicated. (C) OVMz cells were transfected with siRNA specific for ADAM10 or ADAM17. Expression levels were analyzed 48 h after transfection by flow cytometry with mAbs to the ectodomains of ADAM10 or ADAM17. (D) siRNA transfected cells were lysed in Triton X-100 and sucrose gradient centrifugation was carried out as described above. Gradient fractions were analyzed by Western blot with pcytL1 as described above. Numbers indicate relative band intensities. (*: unspecific bands)

Figure 2: L1-32 cofractionates with late endosomal marker LAMP-1

(A) Fraction # 7 of the raft gradient was harvested, diluted with PBS and subjected to further ultracentrifugation for 2 h at 100.000xg. The pellet was suspended in 0.5 ml 40.6 % sucrose and loaded into the bottom of a SW60 tube. This was overlaid with 1.5 ml 35 % sucrose, followed by 1 ml 25 % sucrose and finally 0.5 ml 8% sucrose. The gradient was centrifuged for 1 h at 160.000xg in a Beckman SW60 rotor. 1 ml fractions were collected from the top of the gradient and precipitated with chloroform/methanol. Late endosomes (LE) were enriched at the 8/25 % interface, early endosomes (EE) at the 25%/35% interface and heavy membranes (HM) between 35% and 40.6 %. (B) Gradient fractions were probed with pcytL1 and antibodies to LAMP-1 and rab11. presenilin inhibitor DAPT or the solvent DMSO. Cell lysates were prepared in lysis buffer containing TX-100 and sucrose gradient centrifugation was carried out as described above. Gradient fractions were analyzed by Western blot with pcytL1 followed by peroxidaseconjugated secondary antibodies and ECL detection. (C) OVMz cells were treated for 24 h with the presenilin inhibitor IX (DAPT) or the solvent DMSO and lysates were analyzed with pcytL1. Soluble L1 was precipitated from the tissue culture medium and detected by mAb L1-11A to the ectodomain of L1 followed by peroxidase-conjugated secondary antibodies and ECL detection. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FNIII1 FNIII2 FNIII3 FNIII4 FNIII5 v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v L1-

Biochemical

  Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

For

  the analysis of statistical significance the Student´s t test was used. Biochemical Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625

Biochemical

  Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Biochemical Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 3 :

 3 Figure 3: Presenilin/ -secretase inhibitors block L1-32 processing (A) OVMz cells were subjected to sucrose gradient centrifugation as described in Fig.1 and the distribution of PS1 was determined. (B) OVMz cells were incubated for 24 h with the

  (D) Cells were grown for 48 h at 37°C in the presence of the presenilin DAPT or DMSO. Isolated membranes were incubated for 2 h at 37°C and separated into pellet or supernatant (SN) fractions by ultracentrifugation. Lanes 1 to 2 show membranes isolated from DMSO treated cells (vehicle), lanes 3 and 4 are membrane preparations from cells incubated with DAPT. Numbers indicate relative band intensities. (*: unspecific bands).

Figure 4 :Figure 5 :

 45 Figure 4: Nuclear translocation of L1-ICD(A) Purity of isolated nuclei as revealed by marker protein analysis. (B) L1-ICD in the nucleus. OVMz cells were grown in 10% FCS, nuclei were prepared and analyzed by Western Blot with pcytL1 (lane 1). Isolated nuclei were extracted with 0.42 M NaCl, soluble proteins (lane 2) and insoluble membranes (lane 3) were separated by centrifugation. (C) HEK293 and HEK293-hL1 cells were cultivated in 10% FCS, nuclei were isolated and analyzed by Western blot with pcytL1. (D) Nuclear localization of L1 in CHO-hL1 cells (middle row) and OVMz cells (bottom row). L1 negative CHO cells were used as control (top row). Cells were fixed with 3% paraformaldehyde, permeabilized with methanol (-20°C) and stained with pcytL1 and Alexa488-conjugated anti rabbit IgG. Note that methanol treatment eliminates membranous L1 staining but leaves intact intracellular and nuclear staining. (*: unspecific bands)Figure 5: Inhibitors of metalloproteases and presenilin block L1 nuclear translocation(A) CHO-hL1 cells were treated for 72 h with TAPI-0, DAPT or TAPI-0/DAPT and nuclear localization of L1 was analyzed. Cells were fixed with 3% paraformaldehyde, permeabilized with methanol (-20°C), stained with pcytL1 and Alexa488-conjugated anti rabbit IgG and analyzed by confocal microscopy. Note that methanol treatment abolishes membranous L1 staining but leaves intact intracellular and nuclear staining. (B-D) Quantification of nuclear staining of L1 in CHO-hL1 cells treated with the indicated compounds for 48 or 72 h. For each analysis n=10 nuclei were encircled and the pixel number in the nuclei was determined. CHO-hL1 cells were used due to easier handling in microscopic analysis.

Figure 6 :

 6 Figure 6: Inhibitors of metalloproteases and presenilin block L1-dependent gene regulation (A) HEK293 or HEK293-hL1 cells were treated with DMSO, DAPT, TAPI-0 or both inhibitors for 96 h. RNA was transcribed to cDNA and expression of CRABPII and CathepsinB was analyzed by qRT-PCR. (B) Analysis of ERK phosphorylation in HEK293 and HEK293-hL1 cells treated with the indicated compounds.

Figure 7 :

 7 Figure 7: Targeting of PS1 or ADAM10 blocks L1-dependent gene regulation (A) OVMz cells were transfected with siRNA targeting L1. The knock-down of L1 was analyzed 72 h after transfection using pcytL1 and ECL detection. (B) RNA was transcribed to cDNA and expression of MDK and beta3 integrin was analyzed by qRT-PCR. (C) OVMz cells were transfected with siRNA targeting PS1 or ADAM10. Knock-down was confirmed with antibodies to ADAM10 (mAb 11G2) or PS1. (D) RNA was transcribed to cDNA and expression of MDK and beta3 integrin was analyzed by qRT-PCR. One representative experiment is shown (n=3).

Biochemical

  Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 8 :

 8 Figure 8: A L1-cytoplasmic fragment mediates transcriptional regulation PT45-PI cells were transduced with retroviral vectors encoding L1cyt or full-lenght L1. (A) Expression levels were analyzed by intracellular staining with 745H7 to L1 followed by flow cytometry (B) Western Blot analysis with pcytL1 followed by peroxidase-conjugated secondary antibodies and ECL detection. (C) Detection of L1-ICD in isolated nuclei. Nuclei were prepared and immunoblotted with pcytL1 as described in the legend to Fig. 4. (D) RNA was isolated and transcribed to cDNA. Expression of CRABPII, CathepsinB and beta3 integrin was analyzed by qRT-PCR. One representative experiment is shown (n=3).

  Fig. 1

  Fig.2

  Fig. 8

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081625 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081625 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We thank Natalie Erbe-Hofmann for excellent technical assistance. This work was supported by grants from Deutsche Krebshilfe to P.A. (Schwerpunktprogramm: Invasion and Migration) and the EU-FP6 framework programme OVCAD project nr. PE-14034.

Abbreviations


Martin Gutternigg

Dubravko Rendić

Regina Voglauer

Thomas Iskratsch

Iain B H Wilson
email: iain.wilson@boku.ac.at

MAMMALIAN CELLS CONTAIN A SECOND NUCLEOCYTOPLASMIC HEXOSAMINIDASE Running Title: Mammalian hexosaminidase D

Keywords: N-acetylgalactosaminidase, nucleocytoplasmic, GFP, hexosaminidase, mouse, human

Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterised in recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterise for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability; furthermore, a myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localisation. Transcripts of the corresponding gene are expressed in a number of murine tissues. Based on its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be found from mammalian sources.

Introduction

β-Hexosaminidases (EC 3.2.1.52) are a class of glycoside hydrolases, widespread in nature and present in species from bacteria to man, which remove terminal GalNAc or GlcNAc residues from a range of glycoconjugates such as glycoproteins and glycolipids. It is especially the relationship of human hexosaminidases to lysosomal storage diseases, particularly Tay-Sachs and Sandhoff syndromes [START_REF] Mahuran | Biochemical consequences of mutations causing the GM2 gangliosidoses[END_REF], which has ensured that this class of enzyme is relatively well-studied. In mammals, 'acidic' lysosomal β-hexosaminidases are homo-or heterodimeric enzymes consisting of different combinations of α and β subunits (Hex A, αβ; Hex B, ββ; Hex S, αα); these subunits are encoded by the HEXA and HEXB genes [START_REF] Korneluk | Isolation of cDNA clones coding for the α-subunit of human β-hexosaminidase. Extensive homology between the αand β-subunits and studies on Tay-Sachs disease[END_REF] and are classified by sequence homology as being members of retaining glycoside hydrolase family 20, as defined by the CAZy database [START_REF] Henrissat | New families in the classification of glycosyl hydrolases based on amino acid sequence similarities[END_REF]. Other eukaryotic family 20 enzymes also include plant vacuole hexosaminidases as well as the insect FDL (fused lobes) proteins involved in N-glycan processing [4][START_REF] Léonard | The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing[END_REF][START_REF] Geisler | A fused lobes gene encodes the processing β-N-acetylglucosaminidase in Sf9 cells[END_REF]; this family of enzymes adopts substrateassisted catalysis with a transition state involving an oxazolinium ion [START_REF] Williams | Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state[END_REF]. On the other hand, some β-hexosaminidases follow a different type of catalytic mechanism featuring a covalently-linked glycosyl-enzyme intermediate [START_REF] Vocadlo | Detailed comparative analysis of the catalytic mechanisms of β-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases[END_REF]; these are members of glycoside hydrolase family 3 and are only found in bacteria. Some years ago, a further β-hexosaminidase gene was molecularly identified from mammalian sources; this enzyme, O-GlcNAcase (abbreviated as OGA or NCOAT and encoded by the MGEA5 gene), is defined as a member of glycoside hydrolase family 84 and is predicted to display similarities to family 20 enzymes in terms of its mechanism and its overall protein fold [START_REF] Hurtado-Guerrero | Molecular mechanisms of O-GlcNAcylation[END_REF]10]. The biological role of O-GlcNAcase is to reverse the action of the peptide:N-acetylglucosaminyltransferase (O-GlcNAc transferase; OGT). Both these enzymes are important in intracellular signalling due to their effect on the intracellular balance of Nacetylglucosamine (GlcNAc) and phosphate modifications of nucleocytoplasmic proteins [START_REF] Hart | Cycling of O-linked β-Nacetylglucosamine on nucleocytoplasmic proteins[END_REF][START_REF] O'donnell | Intracellular glycosylation and development[END_REF] and they may indeed associate with each other in vivo [START_REF] Whisenhunt | Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development[END_REF]. Interestingly, though, 90% of the endogenous enzyme is cytoplasmic [START_REF] Wells | Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic β-N-acetylglucosaminidase, O-GlcNAcase[END_REF]; in the case of recombinant forms of O-GlcNAcase, the 130 kDa form is cytoplasmic [START_REF] Gao | Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterisation of a neutral, cytosolic β-N-acetylglucosaminidase from human brain[END_REF] and the 75 kDa form is apparently nuclear [16]. However, there still remained the question as to whether O-GlcNAcase is the only hexosaminidase in the nucleus and cytoplasm.

Suggestive of yet another β-hexosaminidase gene in mammals are some studies on soluble 'neutral' hexosaminidases from animal sources [17][START_REF] Frohwein | Isolation of β-N-acetylhexosaminidase, β-Nacetylglucosaminidase and β-N-acetylgalactosaminidase from calf brain[END_REF][START_REF] Izumi | Neutral β-N-acetylhexosaminidases of rat brain: purification and enzymatic and immunological characterisation[END_REF][20][START_REF] Izumi | Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral β-N-acetylhexosamindases[END_REF]; two types of non-lysosomal activity were found in bovine brain, with different substrate specificities and temperature stabilities, and christened hexosaminidases C and D. Whereas hexosaminidase C is probably identical to the aforementioned O-GlcNAcase [START_REF] Gao | Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterisation of a neutral, cytosolic β-N-acetylglucosaminidase from human brain[END_REF][START_REF] Dong | Purification and characterization of an O-GlcNAc selective N-acetyl-β-D-glucosaminidase from rat spleen cytosol[END_REF], the origin of the hexosaminidase D activity has remained unknown; this latter enzyme has a bias towards N-acetylgalactosaminide substrates and has also been referred to as a neutral N-acetylgalactosaminidase [START_REF] Izumi | Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral β-N-acetylhexosamindases[END_REF]. Recently, we have identified four β-hexosaminidases from Caenorhabditis elegans, which belong to a novel branch of the glycoside hydrolase family 20 [4] and which also have a preference for aryl Nacetylgalactosaminides. Database searching indicated that their closest mammalian relatives are not the lysosomal hexosaminidases, but putatively soluble uncharacterised proteins; thus, our question was whether these novel mammalian genes encoded enzymes with characteristics similar to those of the neutral N-acetylgalactosaminidase. We have, therefore, cloned the relevant human and murine cDNAs and expressed the encoded proteins in Escherichia coli. Indeed, both these proteins are now shown to be enzymatically-active hexosaminidases with properties akin to those of the 'historical' hexosaminidase D activities.

Materials and Methods

Cloning, expression and purification of hexosaminidase homologues

Mouse liver and human embryonic kidney (HEK 293) cells were homogenised in Trizol reagent (Invitrogen, Carlsbad, USA) in order to isolate RNA, which was subsequently subject to reverse transcription using Superscript III (Invitrogen). In order to clone the full predicted open reading frames, the primer pairs MouseHex/1/EcoRI CCGAATTCATGTCATCACCCACGCC and MouseHex/2/XhoI CCGCTCGAGTCAGGGGTTCTGCCCTG or HumanHex/1/EcoRI CGGAATTCATGTCAGGTTCCACTCCATTTC and HumanHex/2/XhoI CCGCTCGAGGCATGAGCTCTCCCCTCA were used together with Expand polymerase mix (Roche) at an annealing temperature of 58 °C in the presence of the GC-rich PCR reaction additive (Roche). After digestion with the relevant enzymes, the fragments were ligated into the pET30a expression vector (Novagen), which encodes a His-tag. Selected transformed E. coli TOP10F´ colonies were screened and the purified plasmids subject to sequencing with the BigDye kit (Applera); subsequently, verified plasmid DNA was used to transform E. coli BL21(DE3)pLysS cells and, after overnight cultivation at 37 °C in LB medium containing kanamycin and chloramphenicol, expression was induced in cultures with OD 600 ~ 0.6 at room temperature in the presence of 1 mM isopropyl-β-D-thiogalactoside (IPTG). In the case of trial expression, aliquots were removed at various timepoints prior to lysis, enzymatic assay and Western blotting with 1:10000 mouse anti-His antibody (HIS-1; Sigma). For larger-scale cultures, lysis and His-tag purification were performed as previously described for GDP-Fuc biosynthesising enzymes [START_REF] Rhomberg | Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster[END_REF]. Peptide mass fingerprinting by MALDI-TOF mass spectrometry was performed after tryptic digestion of SDS-PAGE separated proteins. Gel filtration analysis of the His-tag purified murine hexosaminidase was performed on a Sephacryl S-200 Superfine column equilibrated with 40 mM Tris, pH 7.8, 400 mM NaCl, 100 mM KCl, 250 mM imidazole, 10% glycerol, 0.5% Triton X-100. Approximately 0.75 mg of each of the standard proteins (human IgG, human apo-transferrin, chicken ovalbumin and bovine ribonuclease B) were used to calibrate the column. A modified Lowry protein determination kit (Sigma; TP0300) was used to analyse the fractions; 4 µl aliquots of each fraction were then diluted with 44.75 µl of 0.4 M sodium citrate, pH 5.5, prior to assaying hexosaminidase activity for 1 hr using p-nitrophenyl-β-Nacetylgalactosamine (see below).

Hexosaminidase assays

Samples of crude E. coli lysates (5 µl) or of purified enzymes (0.25-0.5 µl) were assayed with p-nitrophenylglycosides: samples were incubated in microtitre wells with 25 µl McIlvaine citrate-phosphate buffer (pH 3.5-8.0), 1.25 µl of 100 mM p-nitrophenylglycoside in dimethylsulphoxide (final concentrations being 2.5 mM glycoside and 2.5% dimethylsulphoxide) and water (up to a final volume of 50 µl) for 1-2 hr at 37 °C; in some cases, another assay duration, pH or temperature was employed. The reaction was stopped by addition of 250 µl 0.4 M glycine-NaOH, pH 10.4, and the A 405 was read using an microtitre plate reader. The effect of typical hexosaminidase inhibitors were also tested in this study: 2acetamido-1,2-dideoxynojirimycin (2-acetamido-1,2,5-tri-deoxy-1,5-imino-D-glucitol; the kind gift of Dr. Arnold Stütz), N-acetylcastanospermine (6-acetamido-6deoxycastanospermine; Industrial Research Ltd, New Zealand), O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino N-phenylcarbamate (PUGNAc; Toronto Research Chemicals, Canada), streptozotocin (STZ; Sigma), mannosamine (2-amino-2-deoxymannose; Industrial Research Ltd), N-acetylglucosamine and N-acetylgalactosamine (Sigma). For tests with complex substrates, biantennary N-glycans were utilised as before [4], whereas H-Thr-Ala-Pro-Thr-(O-GlcNAc)Ser-Thr-Ile-Ala-Pro-Gly-OH, corresponding to the O-GlcNAc modification site found within the primary sequence of the transcription factor CREB, was purchased from Invitrogen and H-Pro-Gly-Gly-Ser-Thr-Pro-Val-Ser-(O-GlcNAc)Ser-Asn-Met-Met-Ser-Gly-OH, corresponding to a site in casein kinase II, was synthesised by Dr. Nicolas Laurent (University of Manchester).

Tissue-specific expression of murine hexosaminidase

For analysis of tissue specific mRNA expression, adult wild type mice with a mixed B6/129 background were sacrificed; tissues (tibialis anterior, soleus, atrium, ventricle, uterus, lung, liver, kidney and brain cortex) were dissected and immediately frozen at -80 °C until further use. RNA was extracted from tissues using Trizol (Invitrogen) following the manufacturer's instructions. Concentrations of RNA were measured with an UV-spectrophotometer. 2 µg RNA were then mixed with 0.5 µg d(T) 18 and 0.5 µg d(N) 10 primers. Nuclease-free water was added to a final volume of 15 µl. The mix was incubated at 75°C for 5 minutes and subsequently cooled on ice. Then 5 µl of 5× reverse transcriptase buffer, 1.5 µl of 10 mM dNTPs, 0.5 µl RNasin (RNase Inhibitor), 2 µl nuclease-free water and 1 µl of reverse transcriptase (M-MLV, Promega, 100 units/µl) were added. The mix was incubated at 42°C for 90 minutes. Finally the enzyme was denatured by incubating at 75°C for 10 minutes. The resulting cDNAs were normalised against the levels of GAPDH transcripts. The following primer pairs were used which cross intron/exon boundaries: 5MmHexoRT_1 (ATACAGCCCGTCCGAGGTTAC) and 3MmHexoRT_1 (TCAGGGTGTTGGGGAAGAG). PCR was performed for 40 cycles at 98° for 30 sec, 67° for 20 sec and 72° for 15 sec. Furthermore, the primer pair (MouseHex/1/EcoRI and MouseHex/2/XhoI) designed to clone the full open reading frame (see above) was also employed with the various cDNAs using an annealing temperature of 56 °C and an elongation time of 80 sec.

Intracellular localisation

For localisation studies, a vector encoding a N-terminal myc-tag (pSG9M) was employed. Murine liver cDNA was subject to PCR using the primers Mouse/pSG9M/1/XhoI CCGCTCGAGATGTCATCACCCACGCC and Mouse/pSG9M/2/XbaI GCTCTAGATCAGGGGTTCTGCCCTG; the resulting fragments were, after digestion, ligated into the vector; resulting bacterial colonies were screened and plasmid DNA was prepared. Murine 3T3 fibroblasts were transfected using Lipofectin and subsequent to staining with murine anti-myc, followed by FITC or Cy2-labelled anti-mouse IgG, were examined by confocal laser scanning microscopy (Leica TCS SP2 or Zeiss LSM 510) with Ar-laser excitation at 488 nm and emission 500-540 nm or He-Ne-laser excitation at 633 nm and emission > 650 nm using an HC Plans 10×/25 occular and HC PL Fluotar 63×/0.30 or 63x/1.4 objective. Co-labelling with AlexaFluor 633 phalloidin or 4',6-diamidino-2-phenylindole (DAPI) was also performed to detect the cytoskeleton and nucleus, respectively. The cells were also examined by enzymatic assay and Western blotting with mouse anti-myc (9E10, Sigma); in the latter case, a myc-tagged form of Saccharomyces cerevisiae Alg1p β1,4mannosyltransferase expressed in E. coli [24] was used as a positive control.

Results

Identification and expression of novel mammalian hexosaminidase homologues

During our recent characterisation of four novel β-hexosaminidases (HEX-2, HEX-3, HEX-4 and HEX-5) from the nematode Caenorhabditis elegans [4], we became aware of previous uncharacterised homologous human and murine cDNA sequences, which have been annotated in genome databases with the name HEXDC. These two mammalian and four nematode sequences fall into the same subfamily, previously designated by us as 'subfamily 1' [4], of the glycohydrolase family 20 hexosaminidases. This subfamily 1 has been recently described by others, in a bioinformatic and phylogenetic study, as 'clade B' of the glycohydrolase family 20 [25] and their results confirm our previous hypothesis of an evolutionary split in this group of enzymes. Unlike the four nematode genes mentioned above, which encode proteins with predicted transmembrane domains, the two new human and murine cDNAs putatively encode proteins lacking any signal or other hydrophobic sequences and so were considered to possibly encode cytoplasmic proteins. The relevant cDNAs were isolated after RT-PCR as full-length open reading frames and introduced into E. coli expression vectors. In the case of the murine homologue, two different cDNA clones were isolated (one short form and one longer form), whereas in the case of the human one, two identical cDNAs were cloned, both of which lacked an 86 nt region present in the predicted transcript NM_173620, but which are equivalent to the newly-deposited sequence with the Genbank accession number BC018205. The short murine form and the human form, both of 486 amino acids, are encoded by twelve exons and the longer murine form by thirteen exons stretching over some 15-17 kbp of genomic sequence. Only the shorter form of the murine sequence fully corresponds to the human one; the insertion in the longer murine form, encoding an additional 73 amino acids after residue 328, displays no homology to any mammalian sequence and so is presumed to be a splicing artefact. Both the mouse and human proteins contain the His/Asn-Xaa-Gly-Ala/Cys/Gly/Met-Asp-Glu-Ala/Ile/Leu/Val sequence typical of class 20 hexosaminidases (Figure 1); in other members of this family, the glutamate residue of this motif has been shown to be involved in the catalytic mechanism [START_REF] Hou | Characterization of the Glu and Asp residues in the active site of human β-hexosaminidase B[END_REF]. Presumed orthologues of HEXDC sequences occur in other mammals (horse, cow, dog; 78-80% identity with the murine sequence), chicken (63% identity), frog (58% identity) and fish (44% identity) genomes (data not shown). More distantly-related homologues exist in bacteria, such as the StrH β-hexosaminidase from Streptococcus pneumoniae [START_REF] Clarke | Cloning and expression of the β-Nacetylglucosaminidase gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity[END_REF]. Submission of the murine HEXDC sequence to the Phyre server [START_REF] Bennett-Lovsey | Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre[END_REF] indicated that the closest relationships, to proteins with known three-dimensional structure, are to family 20 glycosidases from subfamily 2 (human lysosomal and Streptomyces plicatus hexosaminidases, Actinobacillus actinomycetemcomitans dispersin B and Serratia marcescens chitobiase; E values of 1.8e -32 -1.6e -25 ) and more distantly to family 84 glycosidases (Bacteroides thetaiotaomicron GlcNAcase and Clostridium perfringens NagJ; E values 3e -06 -9.9e -06 ). The long and short murine forms as well as the human HEXDC cDNA were then expressed in BL21(DE3)pLysS cells and, upon induction with IPTG, hexosaminidase activity was detected in lysates of the bacterial cells (Figure 2). Initial indications, when using p-nitrophenyl substrates, were that both enzymes were relatively specific for p-nitrophenyl-β-Nacetylgalactosaminide and that His-tagged proteins of ca. M r 55,000 were expressed in only the induced cells (M r 62,000 in the case of the murine longer form; see Figure 2), as compared to the predicted M r values of 54575 and 53788 for the murine (short) and human proteins. The fact that the longer murine form is active is perhaps due to the insertion in its sequence being outside the region homologous to most other class 20 hexosaminidases of the subfamily 1.

Further characterisation of murine hexosaminidase D

In order to facilitate further characterisation, the enzymes were then partially purified by His-Tag affinity chromatography; the major portions of the activity and anti-His reactivity were in the fractions eluted with 250 mM imidazole. The identity of the purified enzymes was also verified by tryptic peptide mass fingerprinting; due to the 80% identity of the mouse short form and the human form, we performed most experiments with the murine short form, since this was more stable upon storage. Gel filtration analysis of the purified recombinant murine short form suggested that the enzyme is predominantly present as a dimer with a native molecular mass of approximately 120000 (Figure 2C), whereas under denaturing conditions SDS-PAGE and Western blotting demonstrated that a homogenous protein of ca. M r 55000 was present in fractions containing hexosaminidase activity. Bearing in mind the hypothesis that the newly-identified genes encode proteins which may correspond to so-called "hexosaminidase D" activities, the affinity-purified murine enzyme was then examined considering the criteria previously used to categorise the different hexosaminidase activities detected earlier in mammalian tissues [17][START_REF] Frohwein | Isolation of β-N-acetylhexosaminidase, β-Nacetylglucosaminidase and β-N-acetylgalactosaminidase from calf brain[END_REF][START_REF] Izumi | Neutral β-N-acetylhexosaminidases of rat brain: purification and enzymatic and immunological characterisation[END_REF][20]. The apparent relative specificity for p-nitrophenyl-β-N-acetylgalactosaminide (Figure 3A), the pH optimum of 5.5 (Figure 3B) and the relative stability to incubation at 50 °C (Figure 3C) appeared to agree well with previous data on hexosaminidase D [START_REF] Frohwein | Isolation of β-N-acetylhexosaminidase, β-Nacetylglucosaminidase and β-N-acetylgalactosaminidase from calf brain[END_REF][START_REF] Izumi | Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral β-N-acetylhexosamindases[END_REF]. The K m value for the murine enzyme was also determined (~0.25 mM) and was in reasonable agreement to the value of 0.35 mM [START_REF] Frohwein | Isolation of β-N-acetylhexosaminidase, β-Nacetylglucosaminidase and β-N-acetylgalactosaminidase from calf brain[END_REF] also found for the hexosaminidase D (Figure 3D); the temperature optimum was found to be 37 °C (Figure 3E). The pH optimum and relative temperature stability of the human and murine enzymes in E. coli lysates were similar to those of the purified murine enzyme. The effect of various typical hexosaminidase inhibitors was also tested: Nacetylcastanospermine (NACS), 2-acetamido-1,2-dideoxynojirimycin (AdDNJ), O-(2acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc), streptozotocin (STZ), GlcNAc and GalNAc [START_REF] Gao | Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterisation of a neutral, cytosolic β-N-acetylglucosaminidase from human brain[END_REF][START_REF] Dong | Purification and characterization of an O-GlcNAc selective N-acetyl-β-D-glucosaminidase from rat spleen cytosol[END_REF][START_REF] Liu | A potent inhibitor of β-Nacetylglucosaminidases: 6-acetamido-6-deoxycastanospermine[END_REF][START_REF] Gradnig | A novel approach to the 1-deoxynojirimycin system: Synthesis from sucrose of 2-acetamido-1,2-dideoxynojirimycin, as well as some 2-Nmodified derivatives[END_REF][31][START_REF] Lee | The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-β-D-glucosaminidase that contain amino acid residues essential for enzymatic activity[END_REF][START_REF] Gao | Streptozotocin-induced β-cell death is independent of its inhibition of O-GlcNAcase in pancreatic Min6 cells[END_REF][START_REF] Kapur | Purification, biochemical properties and active sites of N-acetyl-β-D-hexosaminidases from human seminal plasma[END_REF]. In the case of our novel murine hexosaminidase, only 60% inhibition with 1 mM PUGNAc and 30% with 1 mM Nacetylcastanospermine was observed when the enzyme was assayed with 2.5 mM substrate (Figure 3F). Thereby, the recombinant murine hexosaminidase D shows properties at variance with O-GlcNAcase, which is inhibited by some 96% by 1.25 mM PUGNAc, when assayed with 2 mM p-nitrophenyl-β-N-acetylglucosaminide [START_REF] Lee | The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-β-D-glucosaminidase that contain amino acid residues essential for enzymatic activity[END_REF]. It also requires far higher concentrations of PUGNAc and N-acetylcastanospermine for inhibition than Arabidopsis HEX1 and Caenorhabditis HEX-1, which are 'typical' (subfamily 2) class 20 hexosaminidases; these two enzymes are inhibited by some 60-90% when incubated with 0.1 mM of these inhibitors under assay conditions similar to those employed here [4]. The relatively poor inhibition of hexosaminidase D with PUGNAc and Nacetylcastanospermine, though, is similar to the results with the p-nitrophenyl-β-Nacetylhexosaminidase activity of four recently-characterised C. elegans class 20 hexosaminidases (HEX-2, -3, -4 and -5), which are part of the same sub-family (sub-family 1) as hexosaminidase D and which also prefer p-nitrophenyl-β-N-acetylgalactosamine as a substrate [4]. Considering the homology of mouse hexosaminidase D with these C. elegans enzymes displaying activity towards N-glycans, the mouse and human hexosaminidases were incubated with N-glycans carrying terminal GlcNAc or GalNAc residues; however, no digestion was observed (data not shown). Furthermore, considering the potential cytosolic localisation of the enzyme, tests were also performed with two peptides modified with Olinked β-GlcNAc; whereas the control reaction with jack bean hexosaminidase showed removal of the GlcNAc residue from the peptides, no such effect occurred in the incubation with the purified murine hexosaminidase (data not shown).

Tissue-specific expression of the novel murine hexosaminidase

RT-PCR was performed using mRNA from a variety of murine tissues (various muscles, uterus, lung, liver, kidney and brain cortex); the cDNAs were normalised based on the levels of GAPDH transcripts and it was found that the putative hexosaminidase D is expressed in all tissues examined, with no major differences in transcript levels (Figure 4).

Intracellular localisation of the novel murine hexosaminidases

Based on the lack of any obvious hydrophobic domains, our hypothesis was that the murine and human forms of the putative hexosaminidase D are not, as other eukaryotic class 20 hexosaminidases, directed via the endoplasmic reticulum to the lysosome or secretory pathway, but that they are present in the nucleus or cytoplasm. Thus, a plasmid encoding an N-terminally myc-tagged form of the full-length murine hexosaminidase was engineered and transfected into murine 3T3 fibroblast cells, which were then subject to immunofluorescence and confocal microscopy. Those cells which were transfected showed that the tagged protein was present in the nucleus and cytoplasm (Figure 5 A and B); it is unclear whether the less elongated appearance of transfected (fluorescent) cells, as compared to the typical fibroblast morphology of the non-transfected ones, is of functional significance. Certainly, actual overexpression of the hexosaminidase was demonstrated by the appearance of a myc-tagged protein of the appropriate molecular mass and of increased N-acetylgalactosaminidase activity in the cell lysates (Figure 5 C andD). In an independent experiment, transfection of COS cells also showed a concentration of the myc-tagged protein in the nucleus (Figure 5 E).

Discussion

A range of earlier literature suggested that, in addition to the well-known lysosomal hexosaminidases encoded by the HEXA and HEXB genes and the neutral cytosolic O-GlcNAcase, there may be an additional soluble hexosaminidase present in mammalian cells. In 1967, a β-N-acetylgalactosaminidase with optimal activity at pH 5.5 was found in a 100,000 g supernatant of a calf brain homogenate [START_REF] Frohwein | Isolation of β-N-acetylhexosaminidase, β-Nacetylglucosaminidase and β-N-acetylgalactosaminidase from calf brain[END_REF]. Later, another report on bovine brain hexosaminidases indicated the presence of four chromatographically-distinguishable enzymes, hexosaminidases A, B, C and D, the latter which was a heat-stable specific galactosaminidase [20]; bovine and human hexosaminidase C, on the other hand, was found as a glucosaminidase with a higher molecular weight and optimal activity in the range pH 5-7 [17, 20, 35] and, in retrospect, probably is identical to O-GlcNAcase [START_REF] Gao | Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterisation of a neutral, cytosolic β-N-acetylglucosaminidase from human brain[END_REF]. Finally, two rat brain neutral hexosaminidases were described which, unlike the lysosomal forms and suggestive of a lack of N-linked glycosylation, did not interact with concanavalin A: one glucosaminidase with, as determined by SDS-PAGE, M r 130000 and one galactosaminidase with M r 55000 [START_REF] Izumi | Neutral β-N-acetylhexosaminidases of rat brain: purification and enzymatic and immunological characterisation[END_REF]. The former apparent molecular weight also corresponds well with that of the rather heat-sensitive O-GlcNAcase; the galactosaminidase, on the other hand, had optimal activity at pH 6 and was relatively heat-stable. In comparison to these data on native enzymes, the recombinant enzymes reported in the present study are soluble, are of M r 55000, are relatively stable at 50 °C, display optimal activity at pH 5.5 and prefer the galactosaminide substrate; thus, it is hypothesised that these human and murine enzymes correspond to the "hexosaminidase D" activities previously detected in mammalian brain extracts. Furthermore, the intracellular localisation data indicates that a myc-tagged form of the murine enzyme is localised to the nucleus and cytoplasm; interestingly, a large-scale yeast two-hybrid screen also suggests that the human enzyme has a high confidence interaction with the tubulin β4 subunit [36]. The recombinant murine enzyme also appears to be predominantly in a dimeric form as is the case with the lysosomal hexosaminidases. Thus, it appears that hexosaminidase D is indeed a second nucleocytoplasmic hexosaminidase with properties, other than the general intracellular location, unlike the O-GlcNAcase proven to play a role in intracellular signalling; on the other hand, 'D-type' hexosaminidases are the only vertebrate members of a recently-described new sub-group within the glycoside hydrolase family 20. Hexosaminidase D does have homologues in nematodes (C. elegans HEX-2, HEX-3, HEX-4 and HEX-5; ca. 30% identity over 440 amino acids) and in insects (Drosophila CG7985; ca. 35% identity), but the predicted proteins contain potential transmembrane regions and so, unlike hexosaminidase D, are not expected to be cytosolic proteins. No function has yet been proven for the insect CG7985, but all four nematode enzymes do digest selected N-glycan substrates in vitro and, as judged by N-glycan analysis of the relevant hex-2 mutant, C. elegans HEX-2 is indeed involved in N-glycan biosynthesis and so is expected to be a Golgi enzyme [4]. Thus, whereas invertebrates possess only the family 84 O-GlcNAcase as a cytosolic enzyme, such as the C. elegans OGA-1 [START_REF] Forsythe | Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism and dauer[END_REF], despite having other family 20 hexosaminidases, the presence of two nucleocytoplasmic hexosaminidases appears to be confined to vertebrates. Akin to the nematode members of the subfamily 1 (clade B) of family 20 and in keeping with the phylogenetic distance [4], the recombinant hexosaminidase D is only inhibited by relatively large inhibitor:substrate ratios when using a range of compounds, such as AdDNJ, NACS or PUGNAc, typically considered to be effective inhibitors of other family 20 hexosaminidases [START_REF] Liu | A potent inhibitor of β-Nacetylglucosaminidases: 6-acetamido-6-deoxycastanospermine[END_REF][START_REF] Gradnig | A novel approach to the 1-deoxynojirimycin system: Synthesis from sucrose of 2-acetamido-1,2-dideoxynojirimycin, as well as some 2-Nmodified derivatives[END_REF][31]. Other than O-linked GlcNAc, there are a variety of N-acetylhexosamine-containing compounds in mammalian cells, including mucin-type O-glycans (α-linked GalNAc linked to Ser/Thr residues), various glycolipids (such as the β-GalNAc-containing ganglioside G M2 , whose catabolism is blocked in Tay-Sachs and Sandhoff syndromes), N-glycans (containing β-linked GlcNAc and, occasionally, GalNAc) and glycosaminoglycans (with both αand βlinked hexosamine-containing repeating units); these, though, are all products of the secretory pathway. In the case of N-glycans, some cytosolic exoglycosidase-mediated degradation does occur, for instance via the action of the Man2c1 α-mannosidase on oligomannosidic glycans derived from misfolded proteins [START_REF] Suzuki | Man2C1, an α-mannosidase is involved in the trimming of free oligosaccharides in the cytosol[END_REF]. It is also noteworthy that mammalian Neu2 sialidases display a similar localisation and pH optimum as does hexosaminidase D [39]; however, unlike hexosaminidase D, Neu2 can cleave sialic acid from N-glycans [START_REF] Ishizuka | Accumulation of free complex-type Nglycans in MKN7 and MKN45 stomach cancer cells[END_REF][START_REF] Tringali | Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules[END_REF] as well as from glycolipids [START_REF] Tringali | Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules[END_REF]. Although, as mentioned above, hexosaminidase D may interact with β-tubulin, a protein shown to carry O-GlcNAc residues [42], we detected no activity towards O-GlcNAc-modified peptides. Thus, it appears that hexosaminidase D may not constitute part of another route for control of the balance between O-GlcNAc and phosphorylation of vertebrate cytosolic and nuclear proteins or it may require another co-factor for such an activity or display a unique substrate specificity. On the other hand, its cytoplasmic localisation would argue against a role in metabolism of N-, O-or lipid-linked oligosaccharides originating from the secretory pathway, although non-classical secretion of this enzyme cannot be ruled out; therefore, the functional and biological significance of this enzyme remains to be resolved.

Figure 1 :

 1 Figure 1: Alignment of hexosaminidase D and other hexosaminidases. The first 331 (of 486) residues of the predicted protein sequences of the murine (short; MmHEXD) and human (HsHexD) forms of hexosamindase D were aligned with the sequences of the four 'subfamily 1' class 20 hexosaminidases from Caenorhabditis elegans (CeHEX-2, -3, -4 and -5), one homologue from Drosophila melanogaster (CG7985; designated here as DmHEX4) and three bacterial homologues (from Clostridium perfringens, Oceanobacillus iheyensis and Streptococcus pneumoniae). The region conserved with respect to 'subfamily 2' class 20 hexosaminidases (e.g., human lysosomal hexosaminidases; also designated as clade A in Ref. 25) is highlighted by asterisks. Residues identical in 9/10 sequences are highlighted in black; those which are conserved in at least 5/10 sequences are shown with a grey background.

Figure 2 :

 2 Figure 2: Recombinant expression of mammalian hexosaminidase D in E. coli. Two clones encoding His-tagged forms of either the short (M1; 486 residues) or long (M2; 559 residues) forms of murine hexosaminidase D and of one clone encoding human hexosaminidase D (H1; 486 residues) were used to transform E. coli and lysates were prepared after cultivation for three hours in the presence (+) or absence (-) of IPTG. (A) Western blotting of transformed E. coli indicates expression of proteins of the expected sizes; (B) assay with either p-nitrophenyl-β-N-acetylgalactosaminide or p-nitrophenyl-β-Nacetylglucosaminide for one hour at 37 °C shows induction of especially p-nitrophenyl-β-Nacetylgalactosaminidase activity with mammalian hexosaminidase D clones. Duplicate data were normalised with 100% corresponding to the activity of the induced cell lysate towards pnitrophenyl-β-N-acetylgalactosaminide in that experiment and the averaged normalised data are from two independent experiments; therefore, each point corresponds to four single enzymatic assays. (C) Gel filtration analysis of the short form of the murine hexosaminidase D (M1) on a Sephacryl S-200 Superfine column calibrated (see insert with scale in terms of M r × 10 -3 ) with IgG (IgG; 150 kDa), apo-transferrin (Tf; 80 kDa), ovalbumin (Ov; 43 kDa), and ribonuclease B (Rn; 15 kDa). Hexosaminidase activity was recorded at 405 nm; the presence of the protein with the expected molecular mass was verified by Western blotting (data not shown).

Figure 3 :

 3 Figure 3: Enzymatic properties of the recombinant purified murine hexosaminidase D. (A) Assays were performed with various p-nitrophenyl αand β-glycosides with 0.25 µl purified hexosaminidase D for 2 hours at 37 °C (pH 5.5); average absorbance values (405 nm) were re-calculated as a percentage of the activity with p-nitrophenyl-β-Nacetylgalactosaminide. (B) pH optimum of purified hexosaminidase D using 2.5 mM pnitrophenyl-β-N-acetylgalactosaminide as substrate (2 hours, 37 °C, McIlvaine buffers); average absorbance values were re-calculated as a percentage of the activity at pH 5.5. (C) Assay of hexosaminidase pre-incubated for 30 minutes at either 4 °C or 50 °C prior to incubation for 2 hours with either 2.5 mM p-nitrophenyl-β-N-acetylgalactosaminide or pnitrophenyl-β-N-acetylglucosaminide at 37 °C; in this experiment the stock solution of the latter substrate was 3 mM in buffer and not 100 mM in dimethylsulphoxide. (D) Hanes' plot of data with various concentrations of p-nitrophenyl-β-N-acetylgalactosaminide as substrate (0.5 µl enzyme, 30 minutes, 37 °C, pH 5.5). (E) Temperature optimum of hexosaminidase D using 2.5 mM p-nitrophenyl-β-N-acetylgalactosaminide as substrate (2 hours, pH 5.5); the data at 50 °C and 70 °C are corrected for values obtained in the absence of enzyme and average absorbance values were re-calculated as a percentage of the activity at 37 °C (pH 5.5). (F) Inhibition of hexosaminidase D using either 0.1 M GalNAc or GlcNAc or 1 mM AdDNJ, STZ, NACS or PUGNAc (0.25 µl enzyme, 2 hours, 37 °C, pH 5.5, 2.5 mM pnitrophenyl-β-N-acetylgalactosaminide); background-corrected absolute absorbance values are presented. In assays with 0.5 mM substrate, the inhibition by 0.2 mM PUGNAc also approached 70% (data not shown). Error bars indicate the standard deviations of assays performed in triplicate or quadruplicate.

Figure 4 :

 4 Figure 4: Distribution of hexosaminidase D transcripts in murine tissues. RT-PCR reactions were performed using cDNAs from various murine tissues which were normalised based on their glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) transcript levels (lower panel); primer sets were used to either detect a small internal fragment (upper panel; ca, 150 bp) or the full length transcript (middle panel; ca. 1600 bp) of murine hexosaminidase D. The data show ubiquitous expression in all tissues examined: tibialis anterior (TA), soleus (Sol), atrium, left ventricle (Ventr), uterus, lung, liver, kidney and brain cortex.

Figure 5 :

 5 Figure 5: Expression of myc-tagged murine hexosaminidase D. A clone encoding a myctagged form of the murine hexosaminidase D (MycHexD) was used to transfect murine 3T3 cells: (A) confocal microscopy of two selected transfected cultures; (B) corresponding antimyc fluorescence micrographs showing nucleocytoplasmic localisation of the construct; (C) comparison of N-acetylglucosaminidase and N-acetylgalactosaminidase activities of nontransfected, 'empty vector' transfected and MycHexD-transfected 3T3 cells showing induction of p-nitrophenyl-β-N-acetylgalactosaminidase activity in the latter; (D) demonstration of expression of MycHexD by Western blotting (lane 1, cells transfected with the vector encoding the myc-tagged form of murine hexosaminidase D; lane 2, cells transfected with 'empty vector'; lane 3, E. coli-expressed myc-tagged β1,4mannosyltransferase positive control). (E) fluorescent micrograph of COS cells transfected with the same MycHexD construct stained with anti-myc (green), DAPI (blue) and phalloidin (red) showing a bias towards nuclear anti-myc labelling; (F) the corresponding blue channel showing DAPI staining alone.

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Acknowledgements

We wish to thank Andreas Rupp, project student in the laboratory, for performing Western blotting, Dr. Boris Ferko for the sample of mouse liver as well as Dr. Elisabeth Ehler for facilitating the work of T.I. on this project, Dr. Johannes Grillari for the pSG9M vector and helpful discussions, Drs. Arnold Stütz and Nicolas Laurent for the kind gifts of chemicallysynthesised compounds and Dr. Katharina Paschinger for reading the manuscript. This work was supported by grants P15475 and P18447 (to I.B.H.W.) from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung. The abbreviations used are: AdDNJ, 2-acetamido-1,2-dideoxynojirimycin; GFP, green fluorescent protein; IPTG, isopropyl β-D-1-thiogalactopyranoside; MALDI-TOF, matrixassisted laser desorption ionisation/time-of flight; MS, mass spectrometry; NACS, Nacetylcastanospermine; OGT, O-GlcNAc transferase; PUGNAc, O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino N-phenylcarbamate; STZ, streptozotocin.

The DNA sequences referred to in this study have been submitted to the Genbank/EBI databank with the numbers FM204885, FM204886 and FM204887.