Eugenia Mato
email: emato@santpau.cat

M Lucas

J Petriz

R Gomis

Anna Novials
email: anovials@clinic.ub.es

Identification of pancreatic stellate cell population with properties of progenitor cells: new

Keywords: SP, Side Population, ABC, ATP Binding Cassette, IAPP, Islet Amyloid Polypeptide, PaSC, Pancreatic Stellate Cells, MET, Mesenchymal-to-Epithelial Transition, HGF, Hepatocyte Growth Factor, BSA, Bovine Serum Albumin, MSC, Multipotent Fibroblast-like Cells, GFAP, Glial Fibrillary acidic protein

Identification of pancreatic stellate cell population with properties of progenitor cells: new role for stellate cells in pancreas

E Mato, M Lucas, J Pétriz, R Gomis, A Novials

To cite this version:

E Mato, M Lucas, J Pétriz, R Gomis, A Novials. Identification of pancreatic stellate cell population with properties of progenitor cells: new role for stellate cells in pancreas. Biochemical Journal, 2009, 421 (2), pp.181-191. [START_REF] Bernard | Neogenesis vs. apoptosis as main components of pancreatic beta cell ass changes in glucose-infused normal and mildly diabetic adult rats[END_REF].1042/BJ20081466. hal-00479072

INTRODUCTION

The Multidrug Resistance Transporter ABCG2 has recently been identified as a molecular determinant of the Side-Population (SP) phenotype, a primitive hematopoietic cell population with a multipotential capacity. This transporter is also expressed in a wide variety of stem cells from adult tissue. It is considered as a novel stem cell marker, and it could be used, therefore, to localize and select these multipotent cells in different tissues [START_REF] Zhou | The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype[END_REF][START_REF] Bunting | ABC Transporters as Phenotypic Markers and Functional Regulators of Stem Cells[END_REF][START_REF] Krishnamurthy | ABCG2/BCRP in Biology and Medicine[END_REF].

In the case of the pancreas, there is no consensus on the nature or localization of adult stem cells [START_REF] Bonner-Weir | Pancreatic stem cells[END_REF][START_REF] Bonner-Weir | The pancreatic ductal epithelium serves as a potential pool of progenitor cells[END_REF]. Several authors have described transdifferentiation in different models in pancreas [START_REF] Von | Islet regeneration needed for overcoming autoimmune destruction -considerations on the pathogenesis of type 1 diabetes[END_REF][START_REF] Gao | In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells[END_REF][START_REF] Rosenberg | Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model[END_REF][START_REF] Rosenberg | Reversal of diabetes by the induction of islet cell neogenesis[END_REF][START_REF] Bernard | Neogenesis vs. apoptosis as main components of pancreatic beta cell ass changes in glucose-infused normal and mildly diabetic adult rats[END_REF]. Also, experimental evidence indicates that the beta cell and other islet cells participate in the intra-islet regeneration [START_REF] Sharma | The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration[END_REF][START_REF] Guz | Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes[END_REF][START_REF] Dor | Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation[END_REF]. Furthermore, a subpopulation of SP cells that co-express ABCG2, MDR1, and Nestin, have been identified and isolated from the adult human pancreas, and they may be a potential source of adult multipotential stem/progenitor cells useful for the production of islet tissue for transplantation into diabetic subjects [14]. However, it is not known whether, in addition to the SP phenotype, other types of pancreatic cells, such as the mesenchymal lineage, also express ABCG2 transporter and represents other adult stem cells that participate in endocrine differentiation.

In order to use adult pancreatic stem cells in potential therapeutic treatment for diabetes, we require more information on the lineage of these cells, the molecular mechanisms that allow them to remain undifferentiated, and the key factors that determine their differentiation [START_REF] Shook | Mechanisms, mechanics and function of epithelialmesenchymal transitions in early development[END_REF]. Pancreatic stellate cells (PaSCs) are associated with diseases such as chronic pancreatitis and fibrosis [START_REF] Phillips | Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover[END_REF][START_REF] Omary | The pancreatic stellate cell: a star on the rise in pancreatic diseases[END_REF]. Their hepatic counterparts have been described as exhibiting other properties, including those of progenitor cells, but in pancreas this role awaits further investigation [18] The aim of this study was identify new ABCG2 (+) pancreatic cell populations and to explore whether they exhibit properties of progenitor cells.

METHODS

Selection of mitoxantrone-resistant cells and differentiation treatments

Fresh pancreata were removed from lactating rats, as approved by the Institutional Animal Care and Use Committee of the IDIBAPS Research Institute, Barcelona, Spain and digested with collagenase to obtain a primary cell culture. The cell digestion was rapidly divided in two homogenous suspension cells, one part was grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco-BRL, Gaithersburg, MD, USA) (25mM of glucose) supplemented with 10% fetal calf serum (FCS), 100 U/ml of penicillin (basal medium) was used as a control culture. The ABCG2 (+) cell line was generated by mitoxantrone (8µM) selection in the rest of the cell suspension. This drug acted through multidrug transporter systems and was diluted in the basal medium described before.

The medium was removed every 2 days and it was gently washed to remove all the dead cells (Stage 1). The cells that survived of mitoxantrone exposition were then placed in fresh DMEM/F12 (11.1mM glucose) (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% FCS. Mitoxantrone-resistant cells have the ability to proliferate until reaching confluence in a monolayer with a fibroblast-like appearance (Stage 2). In the stage 2, several 3D structures known as cell clusters appeared spontaneously, socalled (Stage 3). All the culture was maintained at 37ºC in a humidified atmosphere containing 5% C0

Measurement of apoptotic cell death

To assess drug selection, we analysed apoptosis of cells stained with Hoechst 33342 dye. The bis-benzimidazole dye penetrates the plasma membrane and stains DNA in cells without permeabilization. In contrast with normal cells, the nuclei of apoptotic cells have highly condensed chromatin that is uniformly stained by this dye. These morphological changes may be visualised by fluorescence microscopy. After exposure to mitoxantrone in growth medium, the cells on coverslips were fixed in 4% paraformaldehyde at room temperature for 20 minutes. They were then stained in the incubation buffer for 15 minutes with Hoechst 33342 dye at a concentration of 1 ȝg/ml. The morphological changes were examined under UV illumination using a fluorescence microscope (Olympus IX70-FL, Tokyo, Japan). The dye was excited at 340 nm, and emission was filtered with a 510nm barrier filter. To quantify the apoptotic process, cells with fragmented or condensed DNA and cells with normal DNA were counted. Data shows apoptotic cells as a percentage of total cells.

Flow cytometry

Flow cytometric analysis was performed with a FACScan flow cytometer (BD, San Jose, CA, USA) equipped with an argon-ion laser at 488 nm. Briefly, cells were harvested from the tissue culture flasks following treatment with trypsin. The cell suspension, at a concentration of 1.0 x 10 6 cells/mL, was fixed for 1 hour in 2% paraformaldehyde at 4ºC, washed once in PBS and stained overnight with primary antibodies, as summarized in Table 1. The labelled cells were analyzed on a FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ, USA) by acquisition of 10,000 gated events. Data were stored as listmode files and analyzed with CellQuest (Becton-Dickinson, Franklin Lakes, NJ, USA) and Summit Workstation software.

Drug uptake and retention assays

To allow cell attachment to the surface and to obtain the conditions that would allow optimal growth. The cells were seeded in 12-well tissue culture plates (TPP, Trasadingen, Switzerland) at a final concentration of 1.0 x 10 5 cells/mL, 1mL total volume per well, 24 hours before the dye uptake/retention experiments. In the presence or absence of verapamil (5ȝM), drug uptake was measured by adding the fluorescent substrate (mitoxantrone 8µM) to the culture basal medium for 1 hour at 37°C. After 1 hour of incubation, cells were washed and resuspended in dye-free culture basal medium, and the blocker was maintained to evaluate its effect on dye retention. When dye compatibility was allowed, dead cells were excluded by simultaneous staining with 1 ȝg/mL propidium iodide (stock solution) [START_REF] Garcia-Escarp | Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs[END_REF].

Immunocytochemistry

Cells at distinct stages were plated in 8-well chamber slides (Nalge Nunc International, LAB-TEK). After fixation in 4% paraformaldehyde they were then blocked for 10 minutes at room temperature in 1% BSA and 0.2% saponin, and incubated overnight at 4°C with the primary antibody diluted in blocking solution (Table 1), following standard protocols. The bound antibody was visualised with a fluorescent secondary antibody (Table 1) under a Leica fluorescence microscope. 1% BSA was used instead of primary antibodies for control slides. In differentiation experiments, the total cells were isolated from cellular clusters and then spread on poly-L-lysine-coated slides, using a cytospin (Shandon Inc., Pittsburgh, PA, cytospin 3). The slides were subsequently fixed in ethanol/acetone (1:1vol/vol) for 10 minutes at -20 C and processed by immunocytochemistry techniques.

Oil red O-staining and vitamin A autofluorescence

The Oil red O-treatment for staining intracellular lipid droplets was performed in accordance with a modification of the method described by Lillie [START_REF] Catalano | Elimination of precipitates in oil red O fat stain by adding dextrin[END_REF]. Briefly, cells were fixed with 4% buffered formalin for 10 minutes at room temperature, washed in PBS (twice, one minute each), and stained with a saturated solution of Oil red (Sigma, Taufkirchen, Germany) in acetone: ethanol (1:9) for 45 minutes at room temperature. Subsequently, cells were washed again under flowing cold water for 10 minutes, which resulted in red staining of lipid droplets. Finally, cells were examined by light microscopy.

Vitamin A storage in lipid vesicles was detected by fluorescence microscopy using an excitation wavelength of 320-380nm to visualize the characteristic rapidly fading fluorescent blue-green of this vitamin.

RT-PCR analysis

Total RNA was extracted from several stages of the cultures using RNeasy extraction kit (Quiagen). To remove genomic DNA contamination, DNase I (Invitrogen, Carlsbad, CA, USA) was used, following the manufacturer's protocol. One µ g of total RNA was reverse transcribed in a buffer solution containing 25 nmol/L MgCl2, 100 mmol/L Tris (pH 8.3), 500 mmol/L KCl, RNAguard (39 U/mL; Pharmacia, Uppsala, Sweden), M-MLV-RT (200 U/mL; BRL, Gibco, Uxbridge, UK), 10 mmol/L deoxynucleotide triphosphate(s), and random hexamer priming [d(N6)5'PO4; Pharmacia]. Incubations of 30 minutes at 42ºC, 5 minutes at 94ºC, and 5 minutes at 5ºC were carried out in a total volume of 20 µ L. Complementary DNA (cDNA) was stored at -80ºC until used. All PCRs were performed using 38 cycles. Parallel RT-PCR reactions without reverse transcriptase was performed for each sample. PCR products were visualized with 1 % agarose gel electrophoresis and ethidium bromide staining. The oligonucleotide sequences used for PCR amplification are summarized in Table 2. used in this study are described in Table 2. A standard curve was generated from five serial dilutions of ARIP cell line synthesised cDNA for Pdx-1. Samples were analysed in triplicate, negative controls were included, and PCR products were verified using dissociation curve analysis immediately after RT-PCR. Expression of target gene was determined by normalising to the respective TATA Binding Protein (TBP) levels as a housekeeping gene. Results were analysed using SDS2.1 software (Applided Biosystems).

Real Time-PCR

Infection of Recombinant Adenoviruse (Ngn3)

The adenovirus expressing mouse Neurogenin 3 (Ngn3) and a control adenovirus expressing bacterial ȕ-galactosidase were prepared with the Adeno-X system (Clontech). For viral infection, cells were incubated with adenoviruses at a multiplicity of infection (MOI) of 50 for 2 hours at 37°C. The virus-containing medium was then replaced, and cells were cultured in the basal medium for 48 hours.

Assays for insulin secretion

Insulin secretion was measured by static incubation, as previously described [START_REF] Kawazu | Monolayer culture of human fetal and adult pancreas. Static and dynamic studies of insulin release in vitro[END_REF]. Cells were plated in 12-well plates at 1.0 x 10 6 cells per well or 100 cell clusters (mitoxantrone-resistant cells at stage 3). Cells were pre-incubated for 1 hour in Krebs-Ringer buffer (KRB) and then incubated for 60 minutes at 37ºC in KRB containing 120mM NaCl, 5mM KCl, 2.5mM CaCl 2 , 1.1mM MgCl 2 , 25mM NaHCO 3 , 0.1% bovine serum albumin and glucose at various concentrations. KRB media were collected and stored at -70ºC until they were assayed for insulin. Insulin was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden) that recognizes only mature insulin, following the manufacturer's instructions. This assay has <20% cross-reactivity with pro-insulin.

Electron microscopy

Cell clusters with and without Matrigel were pre-fixed in Karnovsky's fixative (2.5% glutaraldehyde and 2.0% paraformaldehyde) in 0.1 mol/L phosphate buffer (pH 7.3). The tissues were fixed in 1% freshly prepared osmium tetroxide and 0.8% potassium ferrocyanide in double distilled water for 1 hour. After 3 washes in cold double distilled water, the tissue blocks were dehydrated through an ascending concentration of acetone (30%, 50%, 70%, 95%, and 100%), and three changes of 100% acetone. They were then embedded in Spurr resin [START_REF] Spurr | A low viscosity epoxy resin embedding medium for electron microscopy[END_REF] and polymerized at 60°C. The embedded blocks were sectioned using a diamond knife (Diatome) on a Leica Ultracut UCT (Leica Microsystems, Deerfield, IL). Ultrathin sections were placed on a copper grid, and stained with uranyl acetate and lead citrate [START_REF] Reynolds | The use of lead citrate at high pH as an electron opaque stain in electron microscopy[END_REF] before examination under a JEM 010 (Jeol Ltd., Tokio, Japan) equipped with a Gatan/MegaScan model 792 digital camera (Gatan Inc., Pleasanton, CA).

Statistics

Results are expressed as mean (SEM). The Student's t test was used for paired data. A p value of <0.05 was considered significant.

RESULTS

Establishment of ABCG2 (+) pancreatic rat cell culture

Several pancreata of lactating rats were collected and digested with collagenase. The specific drug mitoxantrone was then used for two weeks to select cells. A signal of To determine whether mitoxantrone-resistant cells were able to increase drug accumulation, the intracellular drug concentrations of mitoxantrone were determined by flow cytometry in the unselected cells or after cell selection, described in the material and method, using verapamil as a specific ABCG2 inhibitor. As shown in Fig. 2 A, a significant increase of the intracellular concentration of the drug in the selected cells when the mitoxantrone plus verapamil was used. This increment was 129.8 %, but no significant increase was observed in the unselected cells, which was used as a control cell line. Moreover, gene expression of ABCG2 by RT-PCR confirmed that the primary culture of drug-resistant cells expressed this transporter (Fig. 2B).

Characterization of mitoxantrone-resistant cells

After cell drug selection, several cellular expansions without mitoxantrone were done.

During cell growth, we can observe as three-dimensional structures (cell clusters) were formed in the cultures (Stage 3) with an average diameter of 29.8 µM±11.4 (Fig. 3B a,b,c,d,e). There was a 2.5-fold decrease in the proliferation ratio compared with that of the monolayer (data not shown), which indicated the presence of distinct cellular stages in the same cultures. Positive staining for Nestin and Thy1.1 (the two stem cell markers proposed) was observed in the cell obtained after cell selection and no evidence of positive signals were detected in the unselected cells (Fig. 3A). Moreover, although Cytokeratin 19 (CK19) was negative in all passages studied from mitoxantrone-resistant cells (supplementary Fig. S1 and Fig. S2 A), Nestin and Thy1.1 were positive. However, this positivity decreased in the highest passages from mitoxantrone-resistant cells, without negativization (supplementary S2 C). To confirm these results, gene expression analysis by RT-PCR was done with these markers in unselected cells (control cells) and in mitoxantrone-resistant cells. The results showed that while no signals were detected in unselected cells, the expression in the mitoxantrone-resistant cells was observed in both stage studies, (Fig. 3C). However, when quantification of the progenitor markers (Nestin and Thy1.1) were assessed by flow cytometry, differences were detected between cells growing in mitoxantrone-resistant cells at stage 2 or in mitoxantrone-resistant cells at stage 3 (Nestin 85.97%±10.3 in Stage 2 vs. 29.5%±7.9 in stage 3; Thy1.1 91.3%±3.4 in Stage 2 vs. 12.9%±6.5 in Stage 3), without any significant differences in the mean fluorescence intensity (Fig. S2 C). Moreover, most of the mitoxantrone-resistant cells displayed strong labelling for N-CAM (Fig. 3 A) and also a strong auto-fluorescent signal was observed (Fig 3 A). To examine the phenotype of the mitoxantrone-resistant cell cultures, several markers were studied: vimentin (mesenchymal lineage), desmin (intermediate filament involved in the myogenic differentiation), alfa-actin (smooth muscle actin filament), GFAP (intermediate filament expressed in neural stem cells) and chromogranin A (present in secretory granules of neuroendocrine cells). All these markers were detected in mitoxantrone-resistant cells at stage 2 and stage 3 by immunocytochemistry (Fig. 4 A,B), and confirmed by RT-PCR (Fig. 4 C). However, the expression of these markers was slightly lower at stage 3 than in stage 2 confirmed by flow cytometry (supplementary Fig. S2 C). As cells showed a high auto-fluorescence signal, oil-red staining was performed to identify the presence of liposoluble material in the cell cytoplasm. Positive droplets were detected in the cytoplasm, thus confirming the presence of liposoluble material in the cell cytoplasm compatible with vitamin A in most of the mitoxantrone-resistant cells in our primary culture. In addition, the percentage of oil-red staining observed in the slides was lower in the stage 2 than in the stage 3. Moreover, the characteristic fading fluorescent bluegreen confirmed that these droplets corresponded to vitamin A. The overall characterization of our cultures indicated that the population selected corresponded to pancreatic stellate cells, the origin of which was mesenchymal. Moreover, the cells displayed several degrees of activation, as shown by the different amount of cytoplasmic vitamin A recorded between Stages 2 and 3 (Fig. 4 A,B).

In vitro endocrine differentiation of mitoxantrone-resistant cell (Stage3)

Numerous studies suggest that the culture medium and the soluble factors added to the cultures play an important role in the maturation of different cell types [START_REF] Royer | Culture medium and protein supplementation in the generation and maturation of dendritic cells[END_REF]. The aim was to demonstrate the capacity of these selected cells to differentiate towards endocrine cells by means of media described in previous cell models (see material and methods). Medium 1 was used as a differentiation cocktail for three weeks. The mitoxantrone-resistant cells showed spontaneous differentiation when grown in basal medium DMEM/F12 (11.1mM glucose) with 10% FCS, as indicated by the presence of a weak band of Pdx-1 transcription factor. No difference in this gene was found when the cultures were treated with medium 1 (M 1) (Fig. 5 A) [START_REF] Haixia | Phenotypic Determination and Characterization of Nestin-Positive Precursors Derived from Human Fetal Pancreas[END_REF]. Cultures obtained before cell selection (unselected cells) used as control cells and treated with the same medium (M1) were always negative (Fig. 5 A). However, a weak signal corresponding to the transcript of the neurogenin 3 gene (Ngn3) was observed only in treated mitoxantroneresistant cells with M1 (Fig. 5 A). This gene plays a key role in beta-cell differentiation [START_REF] Schwitzgebel | Expression of neurogenin3 reveals an islet cell precursor population in the pancreas[END_REF]. To verify whether other genes implicated in the endocrine differentiation pathway could be induced in vitro, isolated cell clusters from mitoxantrone-resistant cells at stage 3 were infected with an adenovirus neurogenin 3 construct. After infection, there was an increase in Pdx-1, as well as in other transcription factors that were detected earlier, such as Neuro D and Pax-4, both of which are involved in endocrine cell differentiation (Fig. 5B). Quantitative Real-Time RT-PCR indicated that both media (M1 and M2) increased 3.9-fold±2.9 and 4.9-fold±3.4 respectively Pdx-1 mRNA basal expression (n=3, p<0.05, Fig. 5C). Moreover, the medium 2 (M2) that was supplemented with exendin-4 indicated a higher increase compared with the medium 1 (M1). Exendin-4 is known to produce activity in islet cells in the pancreas via GLP-1 receptors, giving rise to beta-cell activity and insulin release. It also participates in control of glucose and in fat metabolism. In addition, the presence of GLP-1 receptors confirmed by RT-PCR indicates possible effects in these cells (Fig. 5C). Finally, in order to simulate a specific cellular niche for endocrine differentiation, 100 cell clusters were cultured with matrigel as a semisolid medium supplemented with exendin-4 (M 3). After two weeks of culture under these experimental conditions, the transcription of Pdx-1 increased. In addition, a strong insulin signal was detected for the first time. Moreover, IAPP, glucagon, GLUT2 and the convertases PC1/3 and PC2 were also detected after this treatment (Fig 5A). In contrast, expression of the transcription factor p48 and other exocrine genes, such as amylase, were not detected (data not shown). Interestingly, CK 19 expression was observed. Flow cytometry showed that 31.21%±5.2 of cells were c-peptide positive, and 39.54%±2.1 were also positive for Pdx-1, while a few cells were positive for Nestin 0.8%±0.065. In addition, insulin and c-peptide were co-localized in all the cells studied (Fig. 6). We also observed that c-peptide co-localized with vimentin and CK19 in some cells, but not with the other markers studied (alpha-actin and GFPA) (Fig. 6). Moreover, ultrastructural studies showed that the monolayer cells displayed characteristics compatible with activated pancreatic stellate cells: extensive hypertrophy of the rough endoplasmatic reticulum (RER), abundant lysosomes in the cytoplasm, few lipid droplets and the presence of abundant fibers of collagen in the extracellular compartment (Fig. 7A a-d). The clusters that grew in Matrigel plus exendin-4 showed substantial ultrastructural changes, with smaller and homogenous cell size, with round nuclei and electron-dense homogenous chromatin, a significant increase in the number of mitochondria, lipid droplets in their cytoplasm and also abundant electron-dense granules were also observed (Fig. 7A e-h).

Finally, insulin secretions of several sets of cell clusters were measured by static incubation at low (2.8mM) and high (20mM) levels of glucose. Insulin secretory response to high glucose increased by 44% (911.8 ± 31.7pg /cell cluster) vs. low glucose (635.7±13.4pg /cell cluster) (p<0.05) (Fig. 5B). However, insulin secretion in undifferentiated cells obtained during the first expansion phase of the mitoxantroneresistant cell (Stage 2), was below the detection limit for the two glucose treatments tested (Fig. 7B).

DISCUSSION

The therapies currently used to treat diabetes are unsatisfactory, as they do not offer a cure and cannot prevent the development of the complications associated with the disease [START_REF] Shapiro | Strategic opportunities in clinical islet transplantation[END_REF]. The need for new therapeutic strategies, including genetic and cellular approaches, has led researchers to explore alternative cellular sources of insulinproducing cells, such as embryonic stem cells or stem cells isolated from adult tissue [START_REF] Gao | Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture[END_REF]. There is considerable controversy regarding the progenitor of the pancreatic endocrine beta-cell tissue. Ductal, acinar, SP and, very recently, multipotent fibroblastlike cells (MSCs) in the adult human exocrine pancreas have all been proposed as potential progenitors [START_REF] Seeberger | Expansion of mesenchymal stem cells from human pancreatic ductal epithelium[END_REF][START_REF] Weissman | Stem and progenitor cells: origins, phenotypes, lineage commitments, and trans-differentiations[END_REF]. However, the specific signals that induce transdifferentiation, as well as the proliferative signals to expand a specific type of progenitor, must be identified [START_REF] Scharfmann | Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme[END_REF][START_REF] Beattie | Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas[END_REF][START_REF] Kim | Notochord to endoderm signalling is required for pancreas development[END_REF]. Moreover, to identify multipotential cells, specific cell markers are required. The mechanisms controlling islet cell neogenesis are largely unknown. The formation of new islets, as seen from the ductal epithelium, has long been considered one of the mechanisms of normal islet growth after birth and in regeneration and suggests the presence of pancreatic stem cells. It has been documented that there are abundant endocrine progenitor cells in the neonatal pancreas, but little is known about their relative proportions or even phenotypes. Two candidate precursor markers have been proposed: a haematopoietic stem cell marker, c-Kit, and a neural stem cell marker nestin. In a pre-and post-natal rat pancreas, these two markers decreased proportionally with age, coinciding with the appearance and development of endocrine cell types [START_REF] Yashpal | Characterization of c-Kit and nestin expression during islet cell development in the prenatal and postnatal rat pancreas[END_REF]. Multidrug transporter systems, and more specifically, the ABCG2 subtype, have been proposed as a new marker of stem cells. In the human pancreas, this marker has been identified in the primitive hematopoietic stem cells with SP phenotype, located in the islet or around acinar cells. Other pancreatic cells that can express this marker, however, have not yet been investigated [START_REF] Fetsch | Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues[END_REF]. Rats and mice express the ABCG2 gene in several tissues, such as intestine, kidney and testis, but the pancreas has not been investigated to date [START_REF] Tanaka | Tissue distribution and hormonal regulation of the breast cancer resistence protein (Bcrp/Abcg2) in rats and mice[END_REF].

Although the precise physiological function of these transporters in progenitor and differentiated cells is unknown, it has been postulated that they confer protection against a number of xenobiotics, thus maintaining the regenerative capacity of the tissue [START_REF] Leslie | Multidrug resistance proteins: role of Pglycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense (2005)[END_REF].

Here we isolated ABCG2 (+) cells with mesenchymal features from adult pancreatic tissue of lactantig rats. Because of their capacity to adhere and to divide quickly, these cells were maintained in culture, as a cell line, for more than two years. This approach allowed us to characterize these cells. We demonstrate that ABCG2 (+) cells correspond to a population of pancreatic cells known as pancreatic stellate cells. Although their origin is still being debated, they share the characteristics of mesenchymal (vimentin), neural (GFAP) and muscular (desmin and alfa-actin) cells. One of the main characteristics of stellate cells is their capacity to transdifferentiate. When activated, they express alfa-actin and produce many collagen fibers. They thus resemble myofibroblast cells and may, therefore, be involved in tissue repair [START_REF] Apte | Periacinar stellate shaped cells in rat pancreas: identification, isolation and culture[END_REF][START_REF] Kruse | Isolation, long-term culture, and characterization of rat pancreatic fibroblastoid/stellate cells[END_REF]. Recently it has been described that CD133+ hepatic stellate cells exhibit the properties of progenitor cells and display the capacity to develop into endothelial-like and hepatocyte-like cells in vitro [18]. Our study demonstrates that, although stellate cells are not currently considered true stem cells, they share specific markers with other known adult stem cells, such as ABCG2, Nestin, Thy1.1, and N-CAM. The latter participates in signal transduction and in cell type segregation as a mediator of cellular junctions during organogenesis [40].

Although PaSCs do not usually express endocrine genes during cell expansion, spontaneous cell differentiation occurred and these cells showed weak expression of Pdx-1 (a key transcription factor in the endocrine differentiation pathway). Few studies are investigated regarding how culture medium and additional protein components affect the viability and maturation of the cell [START_REF] Royer | Culture medium and protein supplementation in the generation and maturation of dendritic cells[END_REF]. Our results underscore the importance of defining culture medium composition in experimental procedures in order to identify new soluble factors involved in the processes of cellular transdifferentiation. Moreover, to identify instructive signals that induce differentiation during organogenesis, it will be important to determine how such signalling networks are established and how they elicit multiple signalling responses in endodermal cells to activate appropriate genetic programs [START_REF] Ratineau | Endoderm-and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat[END_REF]. Several signalling molecules have been implicated in induction of specific endodermal cell types. However, few of these factors have been examined in adult pancreatic tissue [START_REF] Sttaford | Retinoids signal directly to zebrafish endoderm to specify insulin-expressing beta-cells[END_REF]. Here it is shown that the presence of inductive factors in the extracellular matrix, in addition to other substances participating in pancreatic differentiation, such as exendin-4 (GLP-1 analog), are required to proceed to the transdifferentiation stages in our cellular model. GLP-1 is secreted from the Lcells of the distal ileum and colon; however, numerous studies show that GLP-1 produces an increase in the beta-cell mass by inducing the neogenesis and transdifferentiation of ductal progenitors into islets cells through the expression of Pdx-1 [START_REF] Yue | Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells[END_REF][START_REF] Abraham | Insulinotropic hormone glucagons-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells[END_REF][START_REF] Hui | Glucagon-like peptide 1 induces differentiation of islets duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells[END_REF]. Soluble factors secreted by PaSC are important in pancreas physiology including matrix turnover processes. However, other roles including effects on progenitors have not been investigated. The analysis of PaSC secretome conducted by our group has identified three new proteins: PEDF, LIF and Wnt5b involved in differentiation and developmental processes described in different cellular models [data not shown]. Future experiments will be required to demonstrate that these proteins are able to participate in the spontaneous Pdx-1 expression detected in the culture from mitoxantrone-resistant cells and may also contribute to the high CK 19 expression detected after cell differentiation. In summary we found that mitoxantrone-resistant cells obtained from lactating rats pacreata, express the ABCG2 transporter, has a PaSC phenotype and they are able to secrete insulin after cell differentiation. (A) Nestin, Thy1.1 and N-CAM protein expression was detected by immunostaining in culture from mitoxantrone-selected drug-resistant cells. Autofluorescence was also observed. Hovewer, no expression was observed in cultures from unselected cells, used as a control. The unselected cells were incubated in 1µg/mL DAPI solution for 30 min in the dark. (X20 original magnification). (B) The mitoxantrone-resistant cells became overgrown by cells with a fibroblastoid morphology (a,b). Spontaneously, some cells began to form three-dimensional cell clusters (c,d,e). (C) The immunophenotyping results were confirmed by RT-PCR from one µ g of total RNA of the cells unselected cells (line 1) and mitoxantrone-resistant cells at Stage 2 (line 2) and mitoxantroneresistant cells at Stage 3 (line3). The ARIP cell line was used as a control of the reaction (line 4). The primers were designed across intron(s), when possible, and their sequences and product sizes are listed in the Table 2. The thermal cycle was repeated 38 times in all the genes analysed. RT-PCR analysis of the gene expression profiles was preformed using one µg of total RNA of unselected cells and mitoxantrone-resistant cells. Genes from pancreatic lineage were identified and compared their expression using different media: basal medium (M 0), Medium 1 (M1) and Medium 3 (M 3). Different cell lines were used as a control of the RT-PCR reactions. All primers were designed across intron(s), when possible and their sequences and product sizes are listed in the Table 2

  not shown) and no loss of cellular phenotype was observed. Moreover, to confirm the presence of ABCG2 transporters, functional and expression studies were performed using flow cytometry, immunofluorescence and RT-PCR techniques (see methods below). To induce cell differentiation, three media were tested for 3 weeks: Medium 1; DMEM (24mM glucose) supplemented with 10 ng/mL HGF (R&D Systems, Minneapolis), 0.5 pmol/L betacellulin (R&D Systems, Minneapolis) and 10 mM nicotinamide (Sigma); Medium 2, DMEM/F12 (11.1mM glucose) supplemented with 0.1 nM exendin-4 (Sigma); and Medium 3, DMEM/F12 (11.1mM glucose) supplemented with ITS (Insulin-transferrin-selenium) (Becton Dickinson) plus 0.1nM exendin-4 (Sigma). Matrigel was supplemented in Medium 3 as a base membrane preparation from Engelbreth-Holm-Swarm mouse tumour cells (Becton Dickinson). Matrigel was used following the manufacturer's instructions at a dilution of 1:3 and with one-hour gelling time at 37ºC. Inverted light microscopy was used to monitor morphological changes. In the differentiation experiments, 100 three-dimensional structures obtained from cell passages 12 to 15 were hand picked and dispersed using a solution of trypsin.

  Real time PCR was performed using the double stranded DNA binding dye Power SYBR Green PCR Master Mix using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, California, USA). The sequences of primers Biochemical Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 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

  Biochemical Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 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 apoptosis was observed between days 2 and 4 after drug selection. This signal decreased significantly after the sixth day until its total negativization (day 12 of selection) (Fig1). After this period, several attached cells started to grow forming a monolayer phase with only one cellular phenotype identified as fibroblastoid-like cells (Stage 2) (Fig. 3B a,b).

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 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

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 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

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 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

Fig. 1 .Fig. 2 ABCG2

 12 FIGURE LEGENDS

Fig. 3

 3 Fig. 3 Establishment and Characterization of a Cell Line from mitoxantroneresistant cell population. (A)Nestin, Thy1.1 and N-CAM protein expression was detected by immunostaining in culture from mitoxantrone-selected drug-resistant cells. Autofluorescence was also observed. Hovewer, no expression was observed in cultures from unselected cells, used as a control. The unselected cells were incubated in 1µg/mL DAPI solution for 30 min in the dark. (X20 original magnification). (B) The mitoxantrone-resistant cells became overgrown by cells with a fibroblastoid morphology (a,b). Spontaneously, some cells began to form three-dimensional cell clusters (c,d,e). (C) The immunophenotyping results were confirmed by RT-PCR from one µ g of total RNA of the cells unselected cells (line 1) and mitoxantrone-resistant cells at Stage 2 (line 2) and mitoxantroneresistant cells at Stage 3 (line3). The ARIP cell line was used as a control of the reaction (line 4). The primers were designed across intron(s), when possible, and their sequences and product sizes are listed in the Table2. The thermal cycle was repeated 38 times in all the genes analysed.

Fig. 4

 4 Fig. 4 Mitoxantrone-resistant cells were phenotyped by immunofluorescence and RT-PCR using pancreatic stellate markers.(A) Mitoxantrone-resistant cells at Stage 2 express the markers: alfa-Actin, GFAP, vimentin, desmin, and chromogranin A. To confirm the presence of the vitamin A stored in the fat droplets, oil red staining was performed. (B) Disaggregated from mitoxantrone-resistant cells at stage 3 were immunophenotyped for the same markers, including the oil red staining. Negative controls (Neg) were used. (X20 original magnification). (C) These results were confirmed by RT-PCR using one µ g of total RNA of the mitoxantrone-resistant cells in both stages (stage 2 and stage 3). Control cell lines were used as a control reaction.

Fig. 5

 5 Fig. 5 Pancreatic gene expression profiles (A)RT-PCR analysis of the gene expression profiles was preformed using one µg of total RNA of unselected cells and mitoxantrone-resistant cells. Genes from pancreatic lineage were identified and compared their expression using different media: basal medium (M 0), Medium 1 (M1) and Medium 3 (M 3). Different cell lines were used as a control of the RT-PCR reactions. All primers were designed across intron(s), when possible and their sequences and product sizes are listed in the Table2. The thermal cycle was repeated 38 times in the gene analysis. (B) Effect of adenovirus-mediated extopic expression of ngn3 on key developmental transcription factors and pacneratic marker expression in mitoxantrone-resistant cells at stage Gene profiling expression of: Neuro D, Pax 6, Pax 4, Pdx-1, Nestin, ABCG2, Insulin, Glucagon, IAPP, PC1/3, PC2, CK19, Thy1.1 and N-CAM mRNA were assayed by RT-PCR in mitoxantrone-resistant cells at stage 3 after infection with adenovirus expressiong Ngn3 (AdCMV-ngn3) or as a control of efficiency of infection with a adenovirus expressing ȕ-galactosidase (AdCMV-ҏ ȕ-gal). mRNA was harvested from mitoxantrone-resistant cells at stage 3 24 hours after infection with an adenovirus expressing Ngn3 (AdCMV-ngn3) or as a control adenovirus expressing ȕ-galactosidase (AdCMV-ҏ ȕ-gal). The numbers of PCR cycles were 25 for TBP, 30 for Ngn3, and 38 for the other transcripts. (C) Gene expressions of Pdx-1 and GLP1 receptor were detected by RT-PCR and quantifed their expression by RT-PCR Real-Time in mitoxantroneresistant cell cultures with basal medium (M0), Medium 1 (M1) and Medium 2 (M 2). The Pdx-1 values from cell cultures with M 0 were taken as reference and values were normalised to housekeeping TBP mRNA levels. The bars indicate the SEM of three independent experiments performed in duplicate and * Indicate statistical significance with p<0.05.

  Fig. 5 Pancreatic gene expression profiles (A)RT-PCR analysis of the gene expression profiles was preformed using one µg of total RNA of unselected cells and mitoxantrone-resistant cells. Genes from pancreatic lineage were identified and compared their expression using different media: basal medium (M 0), Medium 1 (M1) and Medium 3 (M 3). Different cell lines were used as a control of the RT-PCR reactions. All primers were designed across intron(s), when possible and their sequences and product sizes are listed in the Table2. The thermal cycle was repeated 38 times in the gene analysis. (B) Effect of adenovirus-mediated extopic expression of ngn3 on key developmental transcription factors and pacneratic marker expression in mitoxantrone-resistant cells at stage Gene profiling expression of: Neuro D, Pax 6, Pax 4, Pdx-1, Nestin, ABCG2, Insulin, Glucagon, IAPP, PC1/3, PC2, CK19, Thy1.1 and N-CAM mRNA were assayed by RT-PCR in mitoxantrone-resistant cells at stage 3 after infection with adenovirus expressiong Ngn3 (AdCMV-ngn3) or as a control of efficiency of infection with a adenovirus expressing ȕ-galactosidase (AdCMV-ҏ ȕ-gal). mRNA was harvested from mitoxantrone-resistant cells at stage 3 24 hours after infection with an adenovirus expressing Ngn3 (AdCMV-ngn3) or as a control adenovirus expressing ȕ-galactosidase (AdCMV-ҏ ȕ-gal). The numbers of PCR cycles were 25 for TBP, 30 for Ngn3, and 38 for the other transcripts. (C) Gene expressions of Pdx-1 and GLP1 receptor were detected by RT-PCR and quantifed their expression by RT-PCR Real-Time in mitoxantroneresistant cell cultures with basal medium (M0), Medium 1 (M1) and Medium 2 (M 2). The Pdx-1 values from cell cultures with M 0 were taken as reference and values were normalised to housekeeping TBP mRNA levels. The bars indicate the SEM of three independent experiments performed in duplicate and * Indicate statistical significance with p<0.05.

Fig. 6

 6 Fig.6The photomicrographs showed a representative images of coimmunolocalization of different markers by cytospin-prepared cells obtained from disagregated cellular clusters after treatment with medium 3. The markers were visualized in red: c-peptide, green: insulin, vimentin, CK19, GFAP, alfa-actin, and yellow as the merges. The MIN-6 cells were used for the immunohistochemistry control. (X40 original magnification (a-l), X20 original magnifications (m-r).

Fig. 7

 7 Fig. 7 Ultrastructural changes and insulin release in the Mitoxantrone-resistant cells at stage 3 after differentiation treatment with medium 3.(A) Transmission electron micrographs of undifferentiated cells (a-d) show high hypertrophy in the rough endoplasmic reticulum (rER), lipid droplets (LD), lysosomes (L) and collagenous fibers (CF). Two types of electron-dense chromatin structure were observed (Ch). However, the differentiated cells (e-h) presented a homogenous size with a round nucleus (N), at times indented, abundant mitochondria (M), and electrondense granules in the cytoplasm were observed (g). (B) Insulin secretion after 1 hour of glucose stimulation at 20 mM vs. 2.8 mM. The results were normalized to 100 cell clusters (n=3) * p< 0.05 (employing Student's t-test).

Table 1 Antibodies for the characterization of the cell culture Anti-rat or human

 1 Dako, Carpinteria, CA, USA Anti-rabbit Cy TM 2 Jackson Immuno Research 711-225-152, anti-guinea pig-AMCA Jackson Immuno Research 706-155-148 and anti-mouse Cy TM 3 Jackson Immuno Research 705-225-147 Biochemical Journal Immediate Publication. Published on 20 Apr 2009 as manuscript BJ20081466 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081466 A c c e p t e d M a n u s c r i p t

		Dilution	Fluorochrome	Source
	Nestin	1/100	Cy TM 2	556309 BD Biosciences Pharmingen,
				Mississauga, CA, USA
	Thy-1.1 (CD90)	1/100	fluoresceine	554897 BD Biosciences Pharmingen,
				Mississauga, CA, USA
	N-CAM	1/100	Cy TM 2	L0015, USBiological, Massachusetts, USA
	GFAP	1/500	Cy TM 2	Chemicon International
	Alfa-Actin	1/10	Cy TM 2	A0760-22 USBiological, Massachusetts, USA
	Vimentin	1/100	Cy TM 2	MAB3400 Chemicon International
	Chromogranin A 1/100	Cy TM 3	RB-9003-PO Bionova, Westinghouse, CA, USA
	Desmin	1/100	Cy TM 2	MCA849 Serotec, Oxford, UK
	C-peptide	1/100	Cy TM 3	4023-01 Linco Research, Missouri,
	Cytokeratin-19	1/1000	Cy TM 2	Z-0622 Dako, Carpinteria, CA, USA
	Insulin	1/2000	AMCA	A0564,

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 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

ACKNOWLEDGEMENTS

The authors thank Scientific and Technical Services of the University of Barcelona (SCT-UB, Campus Casanova) for technical support with electron microscopy, Rosa Gasa for the AdCMV-ngn3 and AdCMV-ȕ-gal constructs, Margarita Nadal for the kariotype assistance and Caroline Newey for editorial assistance.

Authors' affiliations E Mato 1, ¶ , M Lucas 1 , J Petriz 3, # , R Gomis 1,2 , A Novials


Fuensanta W Martínez-Rucobo

Luise Eckhardt-Strelau

Anke C Terwisscha Van Scheltinga

YEAST CHITIN SYNTHASE 2 ACTIVITY IS MODULATED BY PROTEOLYSIS AND PHOSPHORYLATION

Keywords: chitin synthase, overexpression, regulation, posttranslational modification, proteolytic activation, phosphorylation

. The importance of these enzymes for their organisms, together with the lack of chitin synthases in humans, makes them excellent targets for antifungal drugs.

Saccharomyces cerevisiae chitin synthase 2 (Chs2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during logarithmic growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS experiments on purified Chs2 identify twelve phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.

Chitin, a β-1,4-linked polymer of N-acetylglucosamine, is a polysaccharide that has an important structural role in many organisms, such as arthropods, molluscs, cephalopods, and fungi. The amounts of chitin present vary between these organisms; in yeast it constitutes only one percent of the cell wall, but still is an essential component [START_REF] Cabib | Synthesis of the yeast cell wall and its regulation[END_REF][START_REF] Shaw | The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle[END_REF]. Chitin is synthesized from UDP-GlcNAc by chitin synthases (EC 2.4. 1.16). Typically, these enzymes consist of three domains [START_REF] Silverman | Similar and different domains of chitin synthases 1 and 2 of S. cerevisiae: two isozymes with distinct functions[END_REF]: a hydrophilic amino-terminal domain, a highly conserved central catalytic domain and a hydrophobic carboxy-terminal domain integrated into the membrane (Fig. 1). Chitin synthases are classified into glycosyl transferase family 2 [START_REF] Campbell | A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities[END_REF][START_REF] Coutinho | An evolving hierarchical family classification for glycosyltransferases[END_REF], a sequence family that contains a wide range of enzymes. Besides chitin synthases, the most notable examples are the synthases of the structural polysaccharides cellulose, hyaluronan, β-1,3-glucan and β-1,4-mannan. The catalytic domains of the glycosyl transferase family 2 share a GT-A fold [START_REF] Charnock | Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms[END_REF].

Saccharomyces cerevisiae contains three membrane-bound chitin synthases. Each enzyme has its own specific function: Chs1 has a repair function during cell separation [START_REF] Cabib | Chitin Synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae[END_REF], Chs2 is essential for primary septum formation and thus for cell division [START_REF] Silverman | Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae[END_REF], and Chs3 synthesizes the chitin ring at bud emergence, as well as the chitin in the cell wall [START_REF] Shaw | The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle[END_REF]. Since each chitin synthase acts at a different stage of the cell cycle, independent mechanisms for their regulation must exist in the cell. Disruption of specific chitin synthase genes also results in different effects. S. cerevisiae cells lacking Chs1 grow normally, without showing apparent loss of cell wall chitin content [START_REF] Bulawa | The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo[END_REF]. Likewise, disruption of the Chs3 gene was not lethal [START_REF] Valdivieso | CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae[END_REF]. Therefore, these enzymes do not seem to be critical for yeast cell survival. On the other hand, disruption of Chs2 causes severe growth defects and morphological abnormalities [START_REF] Nagahashi | Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites[END_REF][START_REF] Munro | Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity[END_REF]. The regulation of Chs2 is different from that of the other two: its concentration in the cell changes greatly during the cell cycle, in contrast to the constant levels of Chs1 and Chs3 [START_REF] Choi | Are yeast chitin synthases regulated at the transcriptional or the posttranslational level?[END_REF][START_REF] Chuang | Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p[END_REF][15]. So, whereas regulation of Chs1 and Chs3 can only be posttranslational, Chs2 is also significantly regulated by synthesis and degradation.

In this paper we report the heterologous expression and characterization of Chs2. As it had been previously shown that the N-terminal 222 amino acid residues of Chs2 could be truncated without loss of activity or function [START_REF] Ford | Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function[END_REF], we have also overexpressed the active mutant Chs2ΔN222. Biochemical characterization of both proteins allows further insights in Chs2 regulation by posttranslational modification, as well as the role of the N-terminal domain.

Experimental procedures

Bacterial and yeast constructs. Genomic DNA from S. cerevisiae strain Y3437 was used for PCR amplification of the Chs2 and Chs2ΔN222 genes. The primers used for amplifying Chs2 were: 5'-ATTGCACATATGACGAGAAACCCGTTTATGGTGG-3' and 5'-TCCTCGAGGCCCTTTTTGTGGAAAACATTTG-3', containing NdeI and XhoI sites respectively (underlined). For amplification of Chs2ΔN222 the primers were: 5'-ATTGCACATATGGTCTCAGACTTGCCTCCC-3' and 5'-TCCTCGAGGCCCTTTT-TGTGGAAAACATTTG-3', containing NdeI and XhoI sites respectively (underlined). For bacterial expression, the two constructs were inserted into the NdeI and XhoI sites of the pET26b vector (Novagen). For expression in P. pastoris, the constructs were cloned in a modified pPICZ B vector (Invitrogen) containing a Strep 2 tag, prescission cleavage site, multiple cloning site for NdeI and XhoI restriction sites, prescission cleavage site, and His 10 tag; for this system expression is under the control of the alcohol oxidase (AOX1) promoter. All constructs contain a poly-histidine tag for protein detection by Western blot analysis. The correct insertion and sequence of each construct was confirmed by DNA sequencing. P. pastoris culture conditions. The protease-deficient P. pastoris strain SMD1163 (Invitrogen) was used for expression of the recombinant genes. The higher expressing clones were selected according to [START_REF] André | Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen[END_REF] and made into His + mutants by transformation with a pPIC3.5 vector. For expression, SMD1163 cells carrying the foreign gene were grown overnight in MGY medium (0.34 % (w/v) yeast nitrogen base, 1 % (w/v) ammonium sulphate, 1 % (w/v) glycerol, 4 x 10 -5 % (w/v) biotin) to an OD 600 nm of 2-6. The cells were centrifuged at 1,500 x g for 10 min, then suspended to an OD 600 of 1-2 in MM (0.34 % (w/v) yeast nitrogen base, 1 % (w/v) ammonium sulphate, 1.64 µM biotin, 0.5 % (v/v) methanol) and grown for 24 h at 30 °C. Cells were harvested by centrifugation at 1,500 x g for 10 min and the pellets stored at -80 °C or used immediately for further studies. P. pastoris membrane preparation. Cells were suspended to an OD 600 of 50-100 in 30 mM HEPES pH 7.5 and 100 mM NaCl. After addition of an equal volume of ice-cold acid-washed glass beads (0.5 mm diameter), the cells were broken by vortex mixing for eight 30 s bursts separated by 30 s cooling on ice. Glass beads, cell debris and unbroken cells were removed by centrifugation at 1,500 x g for 10 min and the pellet was washed with an equal volume of buffer and centrifuged again. To separate the membrane fraction from the soluble components, the combined supernatants were then centrifuged at 100,000 x g for 30 min. The crude membrane pellet was suspended in 30 mM Tris or HEPES buffer pH 7.0, 7.5 or 8.0 and the protein content determined using the DC protein assay (BioRad).

Bacterial culture conditions. Chs2 and Chs2ΔN222 were expressed in E. coli C41 (Avidis). Bacterial cells were grown at 37 ˚C in TB medium until OD 600 reached 0.6-0.8, and then induced with 2 mM IPTG for 3-5 h. Cells were harvested by centrifugation at 3,300 g for 15 min and used immediately for membrane preparation.

Bacterial membrane preparation. Cells were suspended in 30 mM Tris pH 7.5 and 150 mM NaCl. The cells were then passed three times through a Microfluidiser model M-110L (Microfluidics Corp., Newton, MA) equipped with a 110 µM interaction chamber and a cooling coil, which were immersed in water at 0 °C. After centrifugation at 15,000 g for 10 min, the supernatant was centrifuged at 100,000 g for one hour. The pellet was suspended in 30 mM Tris pH 7.5, and the protein content was determined as above.

S. cerevisiae culture conditions. S. cerevisiae wild type strain YPH419 were grown overnight in YPD medium (1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (w/v) dextrose) to OD 600 0.5 (logarithmic phase) or to OD 600 14 (stationary phase). The P. pastoris membrane preparation protocol was followed, and the supernatant of the 100,000 x g centrifugation step (soluble fraction) was stored at -80 °C or used immediately.

Activity assay. The assay for measuring chitin synthase activity was carried out according to a previously described method [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF][START_REF] Au-Young | Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae[END_REF] in a 50 µl reaction mixture. Unless otherwise indicated, it contained 30 mM Tris pH 7.5, 5 mM MgCl 2 (for Chs2∆N222) or 5 mM MnCl 2 (for Chs2), 32 mM GlcNAc, 1 mM UDP-(U-14 C)-GlcNAc (Amersham, specific activity 553,000 dpm/µmol), and 25 µg of P. pastoris or 800 µg of E. coli membranes. For investigation of the activity at a pH range of 6 to 9, a mixture of buffers was used. This mixture contained 30 mM HEPES, 30 mM MES and 30mM TAPS. To test divalent cation specificity, 5 mM of a cation salt (MgCl 2 , MnCl 2 , CoCl 2 , NiCl 2 or CdCl 2 ) or 5 mM EDTA was used in the reaction mixture. Samples where incubated at 30 ˚C for 30 min for P. pastoris, or 16 to 20 h for E. coli membranes. To measure background activity from the chitin synthases of P. pastoris, membranes that did not contain overexpressed chitin synthases were used. Mean values and errors (standard deviation) were calculated from two sets of three independent experiments, using the program OriginPro 7.5.

Chitin synthesis was stopped by the addition of 1 ml of ice-cooled 10 % trichloroacetic acid, and the insoluble chitin was collected by filtration through a GF/B glass-fibre disc (Whatman) previously soaked in 10 % trichloroacetic acid. The filters were subsequently washed three times with 1 ml ice-cooled 10 % trichloroacetic acid, and twice with 1 ml ice-cooled 66 % ethanol, according to [START_REF] Fähnrich | Improved assay and mechanism of the reaction catalyzed by the chitin synthase from Saccharomyces cerevisiae[END_REF]. Filters were then transferred to scintillation fluid (Rotiscint eco plus, Roth). Synthesized chitin was quantified by determining the radiation level of the filter using a scintillation counter TRI-CARB 1500 (Canberra-Packard).

Proteolytic activation. For activation of Chs2 and Chs2∆N222 by trypsination, P. pastoris membranes were incubated with trypsin (Sigma-Aldrich) for 10 min at 30 ˚C. As previously reported [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF][START_REF] Sudoh | Identification of a novel inhibitor specific to the fungal chitin synthase -Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans[END_REF], the optimal amount of trypsin needed to be ascertained for each batch of membranes. The optimal trypsin/total membranes ratio varied from 1:30 to 1:150. After the 10 min incubation, proteolysis was stopped by adding a two-fold excess 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.

of soybean trypsin inhibitor (Fluka).

To activate Chs2 and Chs2∆N222 using the soluble S. cerevisiae fraction, the soluble fraction was incubated with trypsin for 10 min at 30 ˚C, after which a two-fold excess of trypsin inhibitor was added. This mixture was then added to the chitin synthaseexpressing membranes and incubated for 10 min at 30 ˚C.

Inactivation of soluble S. cerevisiae fraction. The trypsin-activated soluble S. cerevisiae fraction was incubated with each of the following protease inhibitors: pefabloc (100 µM), E-64 (15 µM), leupeptin (400 µM), benzamidin (100 µM) and pepstatin A (20 µM). After this, the mixture was added to Chs2-containing membranes and the activity assay was carried out.

Protein dephosphorylation. 100 µg of P. pastoris membranes were incubated with 2 units of calf intestine alkaline phosphatase (Roche) for 16 h at 4 ˚C.

Partial protein purification. P. pastoris membranes containing overexpressed

Chs2 were diluted in solubilization buffer (200 mM NaCl, 30 mM imidazole pH 7.5, 10 % glycerol (w/v), 1 % Fos-choline-14) plus protease inhibitors (0.1 mg/ml trypsin inhibitor, 1 mM benzamidine, 0.1 mM pefabloc) and incubated for one our at 4 ˚C. The unsolubilized material was removed by centrifugation for one hour at 170,000 g and loaded onto a Ni-sepharose (Amersham) column. The column was washed with wash buffer (70 mM imidazole pH 7.5, 150 mM NaCl, 0.05 % Fos-choline-14) and eluted with buffer containing 300mM imidazole pH 7.5, 150 mM NaCl and 0.05 % Fos-choline-14.

SDS/PAGE: 10% resolving gels were prepared and overlaid with a 3% stacking gel. Before electrophoresis the proteins were denatured in sample buffer (60 mM Tris pH 6.8, 1.5% SDS, 10% glycerol, 0.005% bromphenol blue, 50 mM DTT).

Western blotting. After SDS/PAGE, proteins were transferred onto PVDF membrane (Immobilon TM pore size 0.45 µm, Millipore) using a semi-dry transfer apparatus (Trans-Blot  SD, Bio-Rad). For immuno-detection, proteins were probed with monoclonal anti-poly-histidine antibody (Sigma-Aldrich) and visualized with anti-mouse alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich), both antibodies were diluted 1:1,000 in 20 mM Tris pH 7.5 plus 150 mM NaCl.

Phosphorylation site mapping. The partially purified protein was resolved by SDS/PAGE analysis and the gel fraction containing Chs2 was cut out for phosphopeptide mapping. This analysis was done at the Center for Functional Genomics (University at Albany, Rensselaer, NY). For this purpose, the protein was in-gel tryptic digested and the resulting phosphopeptides were enriched by TiO 2 IMAC. LC-MS/MS was performed on the enriched fraction to identify phosphorylation sites, and on the entire sample to determine the sequence coverage.

Protein phosphorylation. 150 mg of P. pastoris membranes were incubated with 20 units of M phase Cdc2-cyclin B (NEB, recognition motif S/TP-x-K/R) for 30 min at 30 ˚C in a 30 µl reaction mixture containing 200 µM γ-32 P-ATP (Perkin-Elmer, 3 mCi/µmol) and protease inhibitors (0.1mg/ml trypsin inhibitor, 1 mM benzamidine, 0.1 mM pefabloc). After SDS/PAGE, phosphorylated protein was detected by autoradiography.

Results and discussion

We established the heterologous expression of the S. cerevisiae Chs2 and the deletion mutant Chs2ΔN222 in P. pastoris and E. coli. To further study the proteins, we isolated membranes from both expression systems. As observed by immunodetection (Fig. 2), the proteins migrate on SDS/PAGE with the molecular weights expected from their amino acid sequences (115 and 90 kDa for Chs2 and Chs2∆N222, respectively, including affinity tags), showing that neither Chs2 nor Chs2∆N222 are glycosylated. This is consistent with a Chs2∆N193 mutant expressed in S. cerevisia that was shown not to be glycosylated [START_REF] Uchida | Characterization of chitin synthase 2 of saccharomyces cerevisiae II: both full size and processed enzymes are active for chitin synthesis[END_REF], and is further confirmed by the observation that the proteins expressed in E. coli, which lacks the glycosylation machinery of yeast, have molecular weights comparable to the P. pastoris-expressed proteins (Fig. 2).

Chs2 and Chs2ΔN222 expressed in both expression systems are active. However, the activities of E. coli-expressed proteins were significantly lower than those from P. pastoris. Longer incubation time and higher amounts of membranes were needed to measure similar levels of activity. Therefore, for further characterization only the P. pastoris-expressed proteins were used. P. pastoris is a yeast with a chitinous cell wall like S. cerevisiae, and indeed, P. pastoris membranes not expressing Chs2 show chitin synthase activity. This activity is, however, negligible compared to that of overexpressed Chs2 or Chs2ΔN222 (Fig. 3).

To determine whether both Chs2 and Chs2ΔN222 show enzymatic characteristics similar to the wild-type protein, their dependence on both pH and divalent cations was investigated (Fig. 3). The P. pastoris-expressed Chs2 and Chs2∆N222 activities are strictly dependent on divalent cations (Fig. 3b). For Chs2 we obtained the highest activity in the presence of Mn 2+ , and reduced activity with Mg 2+ and Co 2+ . Chs2∆N222 showed high activity in the presence of Mn 2+ and Mg 2+ , with a slight preference for Mg 2+ , and, like the full-length protein, less activity with Ni 2+ and Co 2+ . For all constructs, upon adding EDTA or Cd 2+ to the reaction mixture activity was lost. This preference for Mn 2+ or Mg 2+ fits with X-ray structures of glycosyl transferases with the GT-A fold, which show that either of these two cations bind the two phosphate groups of the UDP-sugar substrate, and are thought to assist in cleaving the sugar-UDP bond [START_REF] Charnock | Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms[END_REF][START_REF] Persson | Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs[END_REF]. The decrease of activity using the various divalent cations matches the decrease of their ionic radii, and also corresponds with the cation selectivity of wild type Chs2 and S. cerevisiae-expressed mutants [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF][START_REF] Uchida | Characterization of chitin synthase 2 of saccharomyces cerevisiae II: both full size and processed enzymes are active for chitin synthesis[END_REF].

Besides the cation dependence, our two constructs also share an optimum pH between 7.5 and 8.5 with the wild-type enzyme (Fig. 3a) [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF][START_REF] Choi | The use of divalent cations and pH for the determination of specific yeast chitin synthetases[END_REF][START_REF] Nagahashi | Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites[END_REF][START_REF] Uchida | Characterization of chitin synthase 2 of saccharomyces cerevisiae II: both full size and processed enzymes are active for chitin synthesis[END_REF]. Together, these results show that in P. pastoris the protein is expressed in a functional form, having the characteristics of the wild type enzyme. Moreover, when treated with appropriate amounts of trypsin, both proteins show high levels of hyperactivation (Fig. 4); this too is consistent with the wild type enzyme [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF][START_REF] Ford | Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function[END_REF]. Interestingly, the two proteins exhibit different levels of proteolytic hyperactivation: 18-fold for Chs2ΔN222 vs. 6-fold for Chs2 (Fig. 4). The increased activation level for Chs2∆N222 as compared to Chs2 implies that the N-terminal domain obstructs trypsin activity, and that its removal could lead to a more exposed activation site.

Regulation of Chs2

Chs2 synthesizes the primary septum separating mother and daughter cell after contraction of the acto-myosin ring [START_REF] Schmidt | In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other[END_REF][START_REF] Tolliday | Direct evidence for a critical role of myosin II in budding yeast cytokinesis and the evolvability of new cytokinetic mechanisms in the absence of myosin II[END_REF], at a very specific time and position. This requires strong regulation of Chs2 activity. Most obviously, the expression and degradation of the enzyme are under tight control, resulting in a half-life of only 25 minutes [START_REF] Chuang | Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p[END_REF]. Although the expression of Chs2 peaks during mitosis [START_REF] Choi | Are yeast chitin synthases regulated at the transcriptional or the posttranslational level?[END_REF], the protein accumulates in the endoplasmic reticulum until mitotic exit, when it is transported to the mother-bud neck [START_REF] Zhang | Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to motherdaughter neck via the secretory pathway in budding yeast[END_REF]. It already disappears from the neck eight minutes later [START_REF] Roh | The septation apparatus, an autonomous system in budding yeast[END_REF][START_REF] Zhang | Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to motherdaughter neck via the secretory pathway in budding yeast[END_REF], to be degraded by the major vacuolar protease PEP4 [START_REF] Chuang | Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p[END_REF].

In addition to this metabolic regulation, post-translational modification has been observed for Chs2. Chs2 is phosphorylated in vitro by, and thus a possible in vivo target of, the cyclin-dependent kinase Cdk1 in complex with the mitotic phase cyclin Clb2 [START_REF] Ubersax | Targets of the cyclin-dependent kinase Cdk1[END_REF][START_REF] Loog | Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates[END_REF]. Also, the in vitro chitin synthase activity is increased markedly by proteolysis [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF].

Proteolytic activation

There is a long-standing question about the putative zymogenic form of chitin synthases. For S. cerevisiae, the three chitin synthases are active in their unprocessed form. Trypsin has been reported to stimulate chitin synthase activity of Chs1 and Chs2 [START_REF] Cabib | The control of morphogenesis: enzymatic mechanism for initiation of septum formation in yeast[END_REF][START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF], but not of Chs3 [START_REF] Choi | The use of divalent cations and pH for the determination of specific yeast chitin synthetases[END_REF]. In the case of Chs1, the stimulation was 7-to 15-fold [START_REF] Cabib | The control of morphogenesis: enzymatic mechanism for initiation of septum formation in yeast[END_REF][START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF], trypsin treatment of Chs2 shows around four times increased activity [START_REF] Sburlati | Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae[END_REF]. In the case of the insect M. sexta, trypsin does not activate chitin synthesis directly. However, trypsin was found to activate a soluble stimulatory factor in the midgut, which increased chitin synthesis by 25-30% [START_REF] Zimoch | Regulation of chitin synthesis in the larval midgut of Manduca sexta[END_REF]. For our P. pastoris-expressed constructs, we measured 6 times activation by trypsination for the full-length protein, and 18 times for the Nterminal deletion mutant (Fig. 4, panel 6).

Even though hyperactivation by proteolysis has been well established for Chs1 and Chs2, no endogenous protease that could take this role has been identified. Chs1 was reported to be activated by S. cerevisiae proteinase B [START_REF] Cabib | The control of morphogenesis: enzymatic mechanism for initiation of septum formation in yeast[END_REF][START_REF] Ulane | The activating system of chitin synthetase from Saccharomyces cerevisiae -purification and properties of activating factor[END_REF]. However, proteinase B did not influence Chs1 activity in vivo [START_REF] Zubenko | Septum formation, cell division, and sporulation in mutants of yeast deficient in proteinase B[END_REF][START_REF] Silverman | Proteinase B is, indeed, not required for chitin synthetase 1 function in Saccharomyces cerevisiae[END_REF], demonstrating that either the observed proteolytic stimulation is not physiologically relevant, or performed by another protease.

We tested the possibility of hyperactivation of Chs2 by factors present in the soluble fraction of S. cerevisiae cells, using cells with a logarithmic growth rate, harvested at an OD 600 of 0.5. We found that incubation with the soluble fraction of S. cerevisiae cells in itself does not influence Chs2 activity in P. pastoris membranes. However, when the soluble fraction is first treated with trypsin, and then after inhibition of trypsin is added to the P. pastoris membranes, it does increase Chs2 and Chs2∆N22 activity two-to four-fold (Fig. 4, panel 3).

A number of controls were necessary to confirm that indeed there was an activating factor in the soluble fraction. First, the trypsin-activated soluble fraction did not show any chitin synthase activity by itself (Fig. 4, panel 4), excluding the possibility that the higher activity is caused by trypsin-activated chitin synthase in the soluble fraction. Second, no hyperactivation was observed when trypsin was inhibited before being added to the membranes (Fig. 4, panel 5), showing that there was no residual Since trypsin activity in the trypsinated soluble fraction has been inhibited prior to adding this mixture to the membranes, the activating factor can only be a part of the soluble S. cerevisiae fraction. Moreover, the need for a protease to trigger the stimulatory effect implies that the activating factor in the soluble fraction is a protein. To test whether this soluble protein could be, like trypsin, a protease, we investigated the effect of a range of protease inhibitors on the activated soluble S. cerevisiae fraction. Before addition to the Chs2-containing membranes, the trypsin-activated soluble S. cerevisiae fraction was incubated with each of the following protease inhibitors: pefabloc, E-64, leupeptin, benzamidin or pepstatin A. Indeed, the addition of leupeptin, a serine and cysteine protease inhibitor, almost completely abolished the stimulatory effect, while it did not affect the activity of Chs2 or Chs2∆N222 by itself (Fig. 5).

Identification and isolation of this yeast protease and genetic experiments as performed by [START_REF] Silverman | Proteinase B is, indeed, not required for chitin synthetase 1 function in Saccharomyces cerevisiae[END_REF] will be necessary to understand the physiological role of this phenomenon. However, some characteristics of the activating protease can already be defined. First, the inhibition of activation by leupeptin shows that the enzyme in question is a serine or cysteine protease. Second, activation was only observed when the soluble S. cerevisiae fraction was extracted from cells growing logarithmically, and not when it was extracted from cells growing in a stationary phase. Therefore, all proteases that are expressed constitutively, as for instance the vacuolar proteases, can be ruled out as the activating protease. Moreover, the timing of expression of this protease hints to the in vivo relevance of proteolytic activation of Chs2. The protease expression is during the logarithmic phase, when cells are dividing, which is consistent with the timing of Chs2 activity: the primary septum is synthesized after mitosis has completed [START_REF] Cabib | The septation apparatus, a chitin-requiring machine in budding yeast[END_REF]. Thus, this protease is expressed during the phase when Chs2 activity would peak.

Interestingly, a very similar type of proteolytic regulation is observed for chitin synthase from M. sexta, where the trypsin-activated serine protease CTLP1 was identified to interact with the chitin synthase [START_REF] Broehan | A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm[END_REF]. Despite this similarity, the location and function of this insect chitin synthase are completely different to those of Chs2: the enzyme creates a protective matrix that lines the midgut of the larvae [START_REF] Merzendorfer | Insect chitin synthases: a review[END_REF]. Similar modes of regulation for two chitin synthases from very different species, performing very different functions, suggest that this activation mechanism might be widespread.

Phosphorylation

Another fast and reversible way of enzyme regulation by post-translational modification is protein phosphorylation. To find out whether phosphorylation influences the activity of Chs2, we submitted P. pastoris membranes containing either Chs2 or Chs2∆N222 to dephosphorylation by the unspecific calf intestine alkaline phosphatase. Dephosphorylation of membranes containing Chs2, whether trypsin-treated or not, does not significantly affect chitin synthase activity (Fig. 6, panel 3 and 4). Therefore, Chs2 does not appear to contain a phosphorylation site that plays a direct role in catalysis. However, when phosphatase treatment is followed by trypsination, activity is almost completely lost (Fig. 6, panel 5), indicating degradation of the protein. These results show that active Chs2 does contain one or more phosphorylation sites. These might, as for glycogen phosphorylase [39], be involved in organization and stabilization of the subunit structure of the enzyme. Similar experiments with Chs2∆N222 show no effect after phosphatase treatment (Fig. 6, panel 5), indicating that the phosphate groups that affect protein stability interact with or are located in the N-terminal domain.

Previous knowledge about phosphorylation of Chs2 is summarized in Fig. 1. So far, Ser82 and Ser86 are the only phosphorylation sites that have been identified for wildtype Chs2 [START_REF] Ficarro | Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae[END_REF]. Additionally, Chs2 has been shown to be an in vitro substrate of the cyclin-dependent kinase Cdk1 [START_REF] Ubersax | Targets of the cyclin-dependent kinase Cdk1[END_REF][START_REF] Loog | Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates[END_REF]. To map the phosphorylation sites of Chs2, we analyzed the purified enzyme by mass spectrometry [START_REF] Gafken | Methodologies for characterizing phosphoproteins by mass spectrometry[END_REF][START_REF] Zheng | Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth[END_REF]. For this type of analysis, microgram amounts of pure protein are required. The level of Chs2 expression in P. pastoris allowed for purification of sufficient amounts of protein. We were able to solubilize the overexpressed enzyme in Fos-choline-14 detergent, and established an IMAC purification protocol using the His 10 affinity tag. This single purification step significantly enriched the Chs2 content in the protein mixture, yielding ~50 % pure Chs2. The enzyme was further separated by SDS/PAGE (Fig. 7a) and could be enriched to 100 % purity for mapping of phosphorylation sites by mass spectrometry.

LC-MS/MS analysis identified twelve phosphorylation sites (Table 1), all in the N-terminal domain. The localization of the sites is consistent with our dephosphorylation experiments. Whereas Chs2 is affected by dephosphorylation, Chs2∆N222, having only one of the twelve sites present, is not. Consequently, we can conclude that dephosphorylation of the N-terminal domain sensitizes the enzyme towards trypsin. Trypsin is a highly unspecific protease, which cleaves a polypeptide chain after almost every exposed positively charged amino acid residue. An elevated sensitivity towards trypsin usually indicates a less compactly folded and thus less stable protein. Dephosphorylation of the N-terminal domain clearly exposes and destabilizes parts of Chs2 that are stable when this domain is phosphorylated or not present at all. This hints at a regulatory function for (de)phosphorylation of the N-terminal domain, as for example an involvement in the short lifetime of the enzyme.

Expression, localization and degradation of Chs2 are cell cycle dependent [15]. The enzyme is mainly expressed during mitosis [START_REF] Choi | Are yeast chitin synthases regulated at the transcriptional or the posttranslational level?[END_REF]. It is transported from the endoplasmic reticulum to the mother-bud neck at mitotic exit, to synthesize the primary septum [START_REF] Zhang | Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to motherdaughter neck via the secretory pathway in budding yeast[END_REF]. Finally, after cytokinesis it is proteolytically processed in the vacuole [START_REF] Chuang | Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p[END_REF]. Such a complex cycle requires tight regulation of activity and stability. If phosphorylation is important for protein stability, the enzyme should be phosphorylated immediately after synthesis, and dephosphorylation could then facilitate rapid degradation of the protein when this becomes necessary.

In this light, it is interesting to note that four phosphorylation sites match the perfect phosphorylation motif (S/TP-x-K/R) for Cdk1, a cyclin-dependent kinase that controls many cell cycle events, and that they are all located in the N-terminal domain (Fig. 1). Furthermore, all four sites are observed in our LC-MS/MS experiments. Indeed, Chs2 has been revealed as an in vitro substrate of Cdk1, with the highest phosphorylation rate when it is complexed with Clb2 [START_REF] Ubersax | Targets of the cyclin-dependent kinase Cdk1[END_REF][START_REF] Loog | Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates[END_REF]. We have used the homologous human complex in combination with γ-32 P-ATP and have shown that Chs2∆N222 is not phosphorylated by this complex while Chs2 is (Fig. 7b). Thus, Cdk1 phosphorylation by Cdk1 is at the N-terminal domain, which is consistent with the phosphorylation sites observed with MS/MS.

The N-terminal domains of chitin synthases can be quite different, and their regulatory role is uncertain [START_REF] Ford | Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function[END_REF]. However, CaChs1, the functional homologue of Chs2 from C. albicans, has an N-terminal domain that is homologous to that of Chs2, and contains three perfect Cdk1 phosphorylation motifs. Indeed, all N-terminal domains that are homologous to that of Chs2 (UniProtKB primary accession numbers Q6FQN5, A7TEI4, Q6CS65, Q758F2 and Q6BZ46) contain multiple perfect Cdk1 phosphorylation sites, in most cases even in the same position as for Chs2. Therefore, it appears that the Cdk1 phosphorylation sites are a feature of this family of N-terminal domains, strengthening the suggestion of a functional role.

Chs2 is most efficiently phosphorylated when Cdk1 is complexed with Clb2 [START_REF] Loog | Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates[END_REF]. As this is an M phase cyclin, it is active during mitosis, when Chs2 is synthesized, but not after cytokinesis, when Chs2 is degraded. Cdk1-Clb2 phosphorylates the N-terminal domain of Chs2, and the timing of its activity is correlated with that of Chs2. As tempting as it is to speculate about a possible role for Cdk1 in regulating Chs2 activity, sitedirected mutagenesis studies will be necessary to confirm whether these specific phosphorylation sites are of importance for the short lifetime of Chs2.

Footnotes -The abbreviations used are: Chs, chitin synthase; GlcNAc, Nacetylglucosamine; MGY, minimal glycerol; OD, optical density; MM, minimal methanol; TB, terrific broth; IPTG, isopropyl β-D-1-thiogalactopyranoside; YPD, yeast extract peptone dextrose; LC, liquid chromatography; MS/MS, tandem mass spectrometry. The table lists the phospho-peptides that were identified based on neutral loss of a mass of 98 and three continuous ion series. The residues denoted with an asterisk (*) are phosphorylated residues identified without ambiguity. The underlined residues are sites identified with neutral loss of a mass of 98 and only two continuous ion series. Sequence coverage was 56%. Fig. 2. Western blot analysis of membranes shows that in both expression systems the recombinant proteins were integrated into the membrane. Each protein migrates according to its sequence-based predicted molecular weight, and to the same level when expressed in yeast or bacteria, which implies that both are not glycosylated in P. pastoris. The His-tagged proteins were probed with monoclonal anti-poly-histidine antibody and visualized with alkaline phosphatase-conjugated secondary antibody. Per lane: M) Marker in kDa, 1) Chs2 (115 kDa) in P. pastoris, 2) Chs2ΔN222 (90 kDa) in P. pastoris 3) Chs2 in E. coli, 4) Chs2ΔN222 in E. coli. To ascertain that all differences measured were due only to pH, for each experiment the same buffer mixture (30 mM HEPES, 30 mM MES and 30 mM TAPS) was used. b. The effect of cations on protein activity, assayed at pH 7.5. Each bar comprises two independent sets of three experiments (n=6). respectively, each set to 100%). 2) Activities of trypsin-treated membranes, 3) activities of membranes treated with alkaline phosphatase, 4) activities of membranes treated with trypsin, followed by alkaline phosphatase 5) activities of membranes treated with alkaline phoshphatase, followed by trypsin. Each bar comprises two independent sets of three experiments (n=6).

Biochemical

  Journal Immediate Publication. Published on 30 Sep 2008 as manuscript BJ20081475 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081475

Biochemical

  Journal Immediate Publication. Published on 30 Sep 2008 as manuscript BJ20081475 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081475A 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 30 Sep 2008 as manuscript BJ20081475 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081475A 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

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Biochemical

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Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 3 .

 3 Fig. 3. Biochemical characterization of P. pastoris-expressed Chs2 and Chs2ΔN222 shows identical enzymatic properties as compared to the wild-type protein. Black and light-grey bars show the activities of Chs2 and Chs2ΔN222, respectively. Dark-grey bars show background activity of native chitin synthases of P. pastoris. a. The dependence of protein activity on pH, in the presence of 5 mM MnCl 2 .To ascertain that all differences measured were due only to pH, for each experiment the same buffer mixture (30 mM HEPES, 30 mM MES and 30 mM TAPS) was used. b. The effect of cations on protein activity, assayed at pH 7.5. Each bar comprises two independent sets of three experiments (n=6).

Fig. 4 .

 4 Fig. 4. Hyperactivation of P. pastoris-expressed Chs2 (black) and Chs2ΔN222 (grey) by trypsin and/or activated soluble fraction of S. cerevisiae. Activities are shown relative to their corresponding 1) base activities (non-treated membranes, 6.0 and 5.5 nmol GlcNAc incorporated for Chs2 and Chs2ΔN222, respectively, each set to 100%). 2) Activities of membranes in the presence of soluble S. cerevisiae fraction, 3) activities of membranes in the presence of trypsin-activated soluble S. cerevisiae fraction, 4) activities of membranes in the presence of inhibited trypsin, 5) activities of trypsin-treated membranes, 6) activity of trypsin-activated soluble S. cerevisiae fraction by itself. Each bar comprises two independent sets of three experiments (n=6).

Fig. 5 .

 5 Fig. 5. Activation of P. pastoris-expressed Chs2 and Chs2∆N222 by the S. cerevisiae soluble fraction is inhibited by leupeptin. Activity of Chs2 (black) and Chs2∆N222 (grey) is shown relative to 1) base activity (non-treated membranes, set to 100%). 2) Membranes treated with activated soluble S. cerevisiae fraction, 3) activated soluble S. cerevisiae fraction inhibited by 400 µM leupeptin before addition to membranes, 4) membranes treated with 400 µM leupeptin. Each bar comprises two independent sets of three experiments (n=6).

Fig. 6 .

 6 Fig. 6. Phosphatase treatment of P. pastoris-expressed Chs2 (black) and Chs2ΔN222 (grey). Activities are shown relative to their corresponding 1) base activities (non-treated membranes, 6.0 and 5.5 nmol GlcNAc incorporated for Chs2 and Chs2ΔN222,

Fig. 7 .

 7 Fig. 7. Phosphorylation analysis. a. SDS/PAGE analysis of partially purified Chs2. Left lane: marker in kDa, right lane: Chs2 (115 kDa) partially purified by IMAC. The single purification step followed by SDS/PAGE allowed for the isolation of pure Chs2 for LC-MS/MS phosphorylation site mapping. b. Autoradiography analysis of an SDS/PAGE gel from P. pastoris membranes containing overexpressed Chs2 or Chs2ΔN222 after treatment with Cdc2-cyclin B plus γ-32 P-ATP. Per lane: M) Marker in kDa, 1) Membranes containing overexpressed Chs2 show a band at the molecular weight of the recombinant protein 2) Membranes containing overexpressed Chs2∆N222 do not show a signal at the molecular weight of the recombinant protein.

Fig. 1 Biochemical

 1 Fig. 1

Fig. 2 Biochemical

 2 Fig. 2

Fig. 3a 3b

 3a Fig. 3a

Fig. 4 Biochemical

 4 Fig. 4

Fig. 5 Biochemical

 5 Fig. 5

Fig. 6 Biochemical

 6 Fig. 6

Fig. 7a 7b

 7a Fig. 7a

Table 1 .

 1 Mass spectrometry identification of phosphorylation sites on Chs2

	4 NPFMVEPS*NGSPNR 17
	4 NPFMVEPSNGS*PNR 17
	36 WANPSEESLEDSYDQSNVFQGLPAS*PSR 63
	64 AALRYSPDRR 73
	93 YAANLQES*PKR 103
	121 DNADLPVDPY*HLSPQQQPSNNLFGSGR 147
	121 DNADLPVDPYHLS*PQQQPSNNLFGSGR 147
	121 DNADLPVDPYHLSPQQQPSNNLFGS*GR 147
	155 YTMSTTSTTAPSLAEADDEKEK 176
	177 YLTS*TTSYDDQSTIFSADTFNETK 200
	177 YLTSTTSYDDQSTIFS*ADTFNETK 200
	254 RNSPEFTEMR 263

© 2008 The Authors Journal compilation © 2008 Biochemical Society

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. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements -We are most grateful to Werner Kühlbrandt for his support and for fruitful discussions, to David Parcej for advice on the Pichia expression system, to Qishan Li for support on the mass spectrometry experiments, and to Paolo Lastrico for help with figures.