Kristin Moreth

Daniel Riester

Christian Hildmann

René Hempel

Dennis Wegener

Andreas Schober

Andreas Schwienhorst

Active site tyrosine is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme

Active site tyrosine is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme

INTRODUCTION

Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are key enzymes in the regulation of gene expression in eukaryotic cells. HATs and HDACs catalyze the addition and the removal of acetyl moieties from the ε-amino groups of lysines near the amino termini of histones, respectively. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are associated with repression of gene expression [START_REF] Grunstein | Histone acetylation in chromatin structure and transcription[END_REF][START_REF] Peterson | HDAC´s at work: everyone doing their part[END_REF][START_REF] Wade | Histone acetylation: chromatin in action[END_REF]. The activity of HATs and, in particular, of HDACs, both affect angiogenesis, cell-cycle arrest, apoptosis, terminal differentiation of different cell types and the pathogenesis of malignant disease [START_REF] Chung | Histone modification: the "next wave" in cancer therapeutics[END_REF]. Not surprisingly, a number of HDAC inhibitors show a potency as promising antitumor agents. Several drug candidates are currently in phase I -III clinical trials [START_REF] Johnstone | Histone-deacetylase inhibitors: novel drugs for the treatment of cancer[END_REF][START_REF] Rosato | Histone deacetylase inhibitors in cancer therapy[END_REF][START_REF] Villar-Garea | Histone deacetylase inhibitors: understanding a new wave of anticancer agents[END_REF][START_REF] Minucci | Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[END_REF].

Four classes of deacetylase enzymes have been categorized on the basis of sequence homology and functional properties [START_REF] Gregoretti | Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis[END_REF]: RPD (reduced potassium dependency)3-like class 1 HDACs, HDA histone deacetylase)1-like class 2 HDACs and class 4 enzymes (HDAC11) are structurally related zinc-dependent enzymes, whereas class 3 enzymes comprise the structurally unrelated NAD-dependent sirtuins [10].

For a long time research on HDACs had focussed mainly on their role in transcriptional regulation. More recently, however, data became available indicating that HDACs may also act on non-histone proteins and small molecules. Examples comprise HDAC 6, which has tubulin deacetylase activity [11][START_REF] Matsuyama | In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation[END_REF][START_REF] Zhang | HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo[END_REF], and bacterial histone deacetylase-like amidohydrolases (HDAHs) [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF][START_REF] Sakurada | Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme[END_REF].

Recently, the crystal structures of two class 1 enzymes, HDLP (histone deacetylase-like protein from A. aeolicus) [START_REF] Finnin | Structures of a histone deacetylase homologue bound to TSA and SAHA inhibitors[END_REF] and HDAC8 [START_REF] Vannini | Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor[END_REF][START_REF] Somoza | Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases[END_REF], as well as that of one class 2 enzyme, FB188

HDAH [START_REF] Nielsen | Crystal structure of a bacterial class 2 histone deacetylase homologue[END_REF] have been solved. Based particularly on enzyme-inhibitor co-complex structures (see for example Figure 3A and B), a mechanism has been proposed which includes features of those from metallo-and serine proteases [START_REF] Finnin | Structures of a histone deacetylase homologue bound to TSA and SAHA inhibitors[END_REF]. By this mechanism (Figure 3C-E), the active site zinc ion would bind to the carbonyl oxygen of the acetyl moiety polarizing the carbonyl group and thereby increase the electrophilicity of the carbon. The zinc ion also binds to the oxygen of a water molecule such that the nucleophilicity of the water oxygen is increased. In analogy to the mechanism of serine proteases, the nucleophilicity of the water molecule is further increased by the negative charge of a buried Asp-His charge-transfer relay system, to which the water molecule is hydrogen bonded. The nucleophilic attack of the water molecule on the carbonyl carbon would lead to a tetrahedral oxyanion transition state which would be stabilized by the aforementioned zinc-oxygen contacts and by a potential hydrogen bond to the hydroxyl group of a tyrosine residue (Y312 in HDAH). Finally, the acetate would be released and the ε-nitrogen of the lysine residue would accept a proton from a second Asp-His charge-transfer-relay system not present in FB188 HDAH or any other class 2 enzymes [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Confirmation of the important role played by the aforementioned active site amino acid residues came from mutagenesis studies [START_REF] Finnin | Structures of a histone deacetylase homologue bound to TSA and SAHA inhibitors[END_REF][START_REF] Nielsen | Crystal structure of a bacterial class 2 histone deacetylase homologue[END_REF][START_REF] Hassig | A role for histone deacetylase activity in HDAC1-mediated transcriptional repression[END_REF]22]. The proposed mechanism has recently been challenged in part by: (i) ab initio calculation studies [START_REF] Vanommeslaeghe | Ab initio study of the binding of trichostatin A (TSA) in the active site of histone deacetylase like protein (HDLP)[END_REF], (ii) experiments with transition state analogue inhibitors designed to mimic the proposed oxyanion intermediate ( 10) and (iii) experiments with substrates containing different acyl leaving groups [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Despite the huge body of data generated throughout many years of HDAC research, relatively little is known about natural substrates of different HDACs. Indeed, studies so far focussed exclusively on amides as possible substrates. In this study, we demonstrate that HDAC enzymes such as HDAH, HDAC1, HDAC3 and HDAC8 also have a very pronounced esterase activity that can be inhibited by known HDAC inhibitors. On the other hand, HDAC inhibitors are also active against known esterases. Specificity towards acyl moieties is similar for both, amidohydrolase and esterase activities. However, mutation of active site tyrosine (Y312 in HDAH) into phenylalanine impairs only amidohydrolase but actually improves esterase activity. Altogether, our experimental results improve our understanding of the catalytic mechanism of HDACs.

Furthermore, we provide first evidence suggesting that at least certain members of the HDAC family may assume the biological role of an esterase.

EXPERIMENTAL

Synthesis of fluorogenic substrates Boc-L-Lys-4-methylcoumarin-7-amide (MCA) and Boc-L-Lys(ε-acetyl)-MCAwere purchased from Bachem (Weil am Rhein, Germany). 4-nitrophenyl acetate and all other reagents for organic synthesis were obtained from Sigma (Taufkirchen, Germany). The ε-Propionylderivative of Boc-L-Lys(ε-acyl)-MCA was synthesized as described [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. The acetyl-and propionyl-esters of 7-hydroxy-4-methylcoumarin were synthesized using standard protocols.

Briefly, 0.5 mmol of 7-hydroxy-4-methylcoumarin was suspended in 2 ml of dioxane. After addition of 90 µl of N-methylmorpholine (NMM) and 180 µl of acetyl chloride (or propionyl chloride) the suspension was incubated for 10 min at room temperature (RT). After addition of

Assays

HDAC1, HDAC3 and N-COR2 were obtained from BPS Bioscience Inc. (San Diego, USA).

HDAC8 was expressed in E.coli and purified as described [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Porcine liver esterase (EC 3.1.1.1.) was from Sigma (Taufkirchen, Germany), Pseudomonas fluorescens lipase was purchased from Fluka (Taufkirchen, Germany). Mutant HDAH genes were generated by the Quik™-Change method using the protocols of the supplier (Stratagene, La Jolla, CA, USA).

FB188 HDAH and its mutants were prepared as described [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF]. For fluorometric assays, all pipetting and fluorescence detection steps were carried out with the help of a robotic workstation (CyBi™-Screen-Machine, CyBio AG, Germany) including a Polarstar fluorescence reader (BMG, Germany). Reactions were conducted in black 96-well microplates.

For assays with chromogenic substrates, a UV-1601 spectrophotometer (Shimadzu, Duisburg, Germany) was used.

Fluorogenic assays.

Amidohydrolase assays were performed similarly to the standard fluorogenic HDAC assay [START_REF] Wegener | A fluorogenic histone deacetylase assay well suited for high-throughput activity screening[END_REF]. Briefly, 10 µl of diluted enzyme solution (corresponding to 50 ng HDAH), 50 µl substrate solution in HD buffer (15 mM Tris-HCl, 250 µM EDTA, 250 mM NaCl, 0.1 % (w/v) PEG8000), pH 8.0, including 50 mM potassium phosphate and 80 µl trypsin solution (1 mg/ml in HD buffer) were mixed and incubated at 30 °C. The release of AMC (7-amino-4-methylcoumarin) was monitored by measuring the fluorescence at 460 nm (λ ex = 390 nm). Fluorescence intensity was calibrated using free AMC. For HDAC1 and HDAC3, the two-step variant of the assay was used. Briefly, 10 µl of diluted enzyme solution and 50 µl substrate solution in HD buffer were mixed and incubated at 30 °C. After 45 min incubation 80 µl trypsin solution were added. After another incubation for 15 min at 30 °C the released AMC was monitored by measuring the fluorescence at 460 nm. KM and Vmax values as well as IC50 concentrations were determined as previously described [25]. Substrate concentrations between 0.5 and 50 µM were used for standard KM and Vmax determinations.

For IC50 determinations a substrate concentration of 2 µM was used.

Fluorescence-based esterase assays were performed by mixing on ice 10 µl of enzyme solution, 180 µl HD buffer, pH8 and 10 µl substrate solution (from a 40 µM stock of either substrate C or D, Figure 1). After switching the reaction temperature to 20 °C the release of HMC (7hydroxy-4-methylcoumarin) was monitored by measuring the fluorescence at 460 nm.

Fluorescence intensity was calibrated using free HMC. Note that the esterase substrates (C and D, Figure 1) showed substantial hydrolysis at higher temperature.

Chromogenic esterase assay.

In the standard chromogenic assay 10 µl of enzyme were mixed on ice with 980 µl HD buffer and 10 µl substrate solution. After switching the reaction temperature to 25 °C the release of 4-nitrophenol was monitored by measuring the absorbance at 405 nm. Absorbance was calibrated using free 4-nitrophenol (ε405 = 18500 M-1 cm-1).

RESULTS

The present study was initiated to examine a possible esterase activity of HDACs. Esters and amides are rather similar in structure and indeed a number of enzymes have been reported to exhibit dual functionalities accepting either substrate classes (e.g. [START_REF] Thompson | Evidence for an extended active center in elastase[END_REF][START_REF] Hui | Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase)[END_REF][28][START_REF] Komeda | A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for -alanyl dipeptides[END_REF]). The work presented herein concentrates particularly on the HDAC-like amidohydrolase (HDAH) from Bordetella(Alcaligenes strain FB188 [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF] as an HDAC homologue which can be easily produced in large quantities. FB188 HDAH exhibits 30% identity over 291 residues with the first domain of human HDAC6 (HDAC6a), 35% identity with the second domain (HDAC6b), and both enzymes share a number of functional similarities. They are zinc-dependent amidohydrolases that are able to efficiently deacetylate chicken histones, and they show the same substrate specificity towards small peptidic substrates [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF][START_REF] Heltweg | Subtype selective substrates for histone deacetylases[END_REF]. In addition, FB188

HDAH is also inhibited by common HDAC inhibitors such as SAHA. Furthermore, the crystal structure of FB188 HDAH reveals the canonical fold of HDACs [START_REF] Nielsen | Crystal structure of a bacterial class 2 histone deacetylase homologue[END_REF].

Histone deacetylase-like amidohydrolase is an efficient esterase. Previously, we have shown that FB188 HDAH is an amidohydrolase that catalyzes the release of acetate from ε-acetylated lysine residues in the context of various substrates including fluorogenic peptides [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF][START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]25,[START_REF] Wegener | Improved fluorogenic histone deacetylase assay for high-throughput-screening applications[END_REF][START_REF] Riester | Profiling of histone deacetylase specificity based on combinatorial fluorogenic substrate libraries[END_REF] as well as eukaryotic histones [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF]. In addition, we demonstrated that the enzyme also converted nonpeptidic amides such cis-(+/-)-N-[4-(hydroxymethyl)cyclopent-2enyl] acetamide [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF]. For Boc-L-Lys(ε-acyl)-MCA substrates (Figure 1, A and B) the acetyl derivative was the superior substrate with KM and Vmax values of 14.4 ± 3.0 µM and 136 ± 22 pmol sec-1 mg-1, respectively [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Boc-L-Lys(ε-propionyl)-MCA, however, did not show a significant conversion [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. To test the possibility that FB188 HDAH also could act as an esterase we studied esterase activity in comparison to that of porcine liver esterase and Pseudomonas fluorescens lipase using 4-methylcoumarin-7-acetate as a substrate (Figure 1,C; Table 1). Cleavage of this substrate resulted in the release of fluorescent 7-hydroxy-4-methylcoumarin, which was monitored by measuring the fluorescence at λ em = 460 nm and λ ex = 390 nm. HDAH indeed proved to exhibit specific esterase activity that is comparable or even superior to those of porcine liver esterase and Pseudomonas fluorescens lipase (Table 1), which were used as controls. For 4-methylcoumarin-7-acetate KM and Vmax values of 0.90 ± 0.13 µM and 14100 ± 2100 pmol sec-1 mg-1 were measured, respectively. For porcine liver esterase the corresponding values were 2.84 ± 0.84 µM and 2150 ± 630 pmol sec-1 mg-1. For

Pseudomonas fluorescens lipase we obtained KM and Vmax values of 1.66 ± 0.30 µM and 183 ± 41 pmol sec-1 mg-1, respectively.

Esterase activity is dependent on the acyl moiety. In the aforementioned experiments with amide substrates FB188 HDAH exhibited a clear preference for acetamides over the corresponding propionyl derivatives. We therefore also tested 4-methylcoumarin-7-propionate as a substrate (Table 1). For the latter substrate KM and Vmax values of 31.2 ± 3.7 µM and 2220 ± 260 pmol sec-1 mg-1 were measured, respectively. Thus, the enzyme again showed a preference for the acetyl derivative, reflected by both a 34-fold increase in KM and a 6-fold decrease in Vmax for 4-methylcoumarin-7-propionate.

Esterase activity is modulated by the nature of the alcoholic moiety. To show if the alcohol moiety of the ester has a significant influence on the kinetic parameters of the ester substrates we compared the conversions of the aforementioned 4-methylcoumarin-7-acetate and 4-nitrophenyl acetate (Figure 1, E; Table 1). For the latter, we measured KM and Vmax values of 11.8 ± 1.8 µM and 33000 ± 4900 pmol sec-1 mg-1, respectively, indicating a 13-fold increase in KM and a 2-fold increase in Vmax.

Esterase activity can be blocked by HDAC inhibitors.

To confirm that the esterase activity resides in the same active site of FB188 HDAH that is used to catalyze amidohydrolase reactions we tested esterase activity in the presence of known HDAC inhibitors (Table 2). For SAHA and CypX IC50 values of 0.43 µM, 0.59 µM were obtained using 4-methylcoumarin-7acetate as a substrate. Comparing these IC50 values to that obtained in the fluorogenic HDAC assay ( [START_REF] Wegener | A fluorogenic histone deacetylase assay well suited for high-throughput activity screening[END_REF]; Table 2) it appeared that the inhibition of amidohydrolase and esterase activities were comparable for both hydroxamate inhibitors.

Interestingly, HDAC inhibitors also showed a significant inhibition of control esterases. For Pseudomonas fluorescens lipase SAHA showed an IC50 values of 3.82. For porcine liver esterase no inhibitory activity was observed for the hydroxamic acids (SAHA, CypX). Here, however, MS275 proved to be a rather good inhibitor (data not shown). Furthermore, for a trifluoromethylketone derivative which we recently had identified in a screening program as a submicromolar inhibitor of HDAH and eukaryotic HDACs, we also could detect submicromolar inhibition of porcine liver esterase (data not shown).

Y312F mutation impairs amidohydrolase but not esterase-activity.

To further confirm that both amidohydrolase and esterase activities reside in the same active site and to study possible similarities in the catalytic mechanism between FB188 HDAH acting as amidohydrolase or esterase we analyzed the effect of mutations on enzyme activity (Table 1).

Whereas mutant H142N (inner charge transfer relay system) proved to be inactive for both enzyme activities, Y312F only decreased amidohydrolase activity. Surprisingly, esterase activity was even improved in the mutant as compared to the wild-type enzyme. Using 4methylcoumarin-7-acetate as a substrate KM and Vmax values of 1.68 ± 0.32 µM and 513000 ± 97700 pmol sec-1 mg-1, respectively, were measured. This is less than a 2-fold increase in KM but a 36-fold increase in Vmax compared to the wild-type enzyme. For 4-methylcoumarin-7-propionate as a substrate KM and Vmax values of 0.22 ± 0.02 µM and 52400 ± 4700 pmol sec-1 mg-1were measured, respectively. This is a 4-fold decrease in KM and a 23-fold increase in Vmax as compared to the wild-type enzyme and the same substrate.

Human HDAC1, HDAC3 and HDAC8 also exhibit esterase-activity. Next, we were interested to know whether human HDACs are also capable, in principle, of executing esterase activity. For (recombinant) human HDAC1, 3 and 8 substrate conversions in pmol/min were compared using the same enzyme and substrate concentrations but different substrates. In Figure 2 the relative substrate conversions are depicted by arbitrarily setting the conversion of the HDAC substrate (Boc-L-Lys(ε-acetyl)-MCA) to 1. Using both 4-methylcoumarin-7-acetate and 4-methylcoumarin-7-propionate as substrates, we clearly demonstrated that HDAC1, HDAC3/N-COR2 and HDAC8 also have esterase activities. However, different specificities concerning the acyl moiety were observed. HDAC1 appeared to have a preference for the propionate derivative of the ester. As compared to Boc-L-Lys(ε-acetyl)-MCA the ester substrates were more efficiently converted by a factor of 3 and 43 for the acetate and the propionate derivative, respectively. For HDAC3/N-COR2 and HDAC8, similar to FB188 HDAH, a preference for the acetate derivative of the ester was observed. Here, as compared to the conversion of Boc-L-Lys(ε-acetyl)-MCA the ester substrates were more efficiently converted by a factor of 48 and 20 for the acetate and the propionate derivative, respectively.

DISCUSSION

Esterase activity of FB188 HDAH. The major biological function of histone deacetylases is believed to be the deacetylation of ε-acetylated lysines in histones and other proteins [START_REF] Kouzarides | Acetylation: a regulatory modification to rival phosphorylation?[END_REF]. For their bacterial counterparts the biological role is much less clear. Experimental data available so far supports the idea that these enzymes may also function as deacetylases of proteins with εacetylated lysine residues [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF] as well as polyamines [START_REF] Sakurada | Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme[END_REF]. Here we present experimental evidences that histone deacetylase-like enzymes are also very catalytically efficient esterases.

Using chromogenic and fluorogenic substrates we demonstrated that FB188 HDAH indeed has esterase activity comparable or superior to that of well-known lipases and esterases. In terms of specific substrate conversion, the catalytic properties of HDAC-like enzymes conform to the general reactivity of amides and esters: in contrast to esters, amides are known to be resonancestabilized as reflected by a more than 103 times slower reaction in OH--catalyzed reactions [START_REF] Bender | Spectrophotometric investigation of the mechanism of α-chymotrypsin catalysed hydrolases[END_REF][START_REF] Zerner | Kinetic evidence for the formation of acyl-enzyme intermediates in the α-chymotrypsin-catalyzed hydrolyses of specific substrates[END_REF][36]. In our study we observed that the bacterial FB188 HDAH hydrolyzed specific ester substrates with deacylation rates that exceeded those of amides by up to 2 orders of magnitude.

Other researchers have reported on similar results obtained with certain serine proteases which also show amidohydrolase and esterase activity [START_REF] Bender | Spectrophotometric investigation of the mechanism of α-chymotrypsin catalysed hydrolases[END_REF][START_REF] Zerner | Kinetic evidence for the formation of acyl-enzyme intermediates in the α-chymotrypsin-catalyzed hydrolyses of specific substrates[END_REF][36][37][38].

In both ester and amide substrates, FB188 HDAH has as significant preference for acetyl over propionyl moieties presumably due to space limitations in the active site cleft [25]. In line with

this assumption the larger 4-methylcoumarin-7-propionate is only bound well in the larger active site of the Y312F mutant, as reflected by a 142-fold decrease in the KM value. On the other hand, comparing substrates based on 4-nitrophenol or 7-hydroxy-4-methyl coumarin it appears that enzyme activity is less dependent on the alcohol moiety. However, as shown for amide substrates [START_REF] Hildmann | A new amidohydrolase from Bordetella/Alcaligenes strain FB188 with similarities to histone deacetylases[END_REF][START_REF] Finnin | Structures of a histone deacetylase homologue bound to TSA and SAHA inhibitors[END_REF], it cannot be excluded that there may be more dramatic effects with other alcohol moieties.

Implications for the catalytic mechanism. Since standard HDAC/HDAH inhibitors such as SAHA and CypX inhibited not only amidohydrolase but also esterase activity, we tend to believe that both catalytic activities reside in the same active site cleft. Although differences between IC50 values measured in different assays in general can be fairly large [39], for SAHA and CypX amidohydrolase and esterase assays the IC50 data differed only by a factor of 2. In addition, both the amidohydrolase and the esterase activities show the same preference concerning acyl moieties, thus arguing against the possibility of two independent and rather different mechanisms. The view that both catalytic activities reside in the same active site gained further support from the fact that a H142N mutation in the "inner charge transfer relay system" was detrimental to both, amidohydrolase activity and esterase function.

In consistency with the aforementioned facts we like to propose a mechanism for ester cleavage (Figure 3C-E) that very much resembles the proposed mechanism for amide cleavage. In both cases a water molecule as the attacking nucleophile is activated by interaction with an Asp-His142 charge-transfer relay system and a zinc ion (Figure 3C). To increase the electrophilicity of the carbonyl carbon the carbonyl bond of the ester or amide is polarized by interactions of the carbonyl oxygen with the zinc ion and with the hydroxyl group of Tyr312 (Figure 3C). Given the higher reactivity of esters compared to that of amides it seems plausible to assume that part of this carbonyl polarization is dispensable in the case of ester cleavage. This would explain the fact that a Y312F mutation did impair the energetically more demanding amidohydrolase activity but still allowed the enzyme to work as an esterase. Furthermore, in the originally proposed mechanism it was stressed that tyrosine would stabilize the tetrahedral oxyanion transition-state by hydrogen bonding to the carbonyl oxygen (Figure 3D). At least in the case of ester cleavage this function of tyrosine is obviously also non-essential. Surprisingly, however, a Y312F mutation did even stimulate esterase activity, particularly Vmax. As pointed out earlier, FB188

HDAH shows space limitations in the active site cleft. These structural constraints may force the substrate and side chains of the enzyme into unfavorable conformations. In line with this assumption, the absence of a hydroxyl group in mutant Y312F would provide the active site with additional space, i.e. conformational freedom that could stimulate catalytic activity at least in the case of ester cleavage. Future crystallographic studies have to specify the structural differences between wild-type HDAH and the Y312F mutant that presumably causes the observed differences in catalytic activity.

The fact that amidohydrolase activity is more sensitive to small structural perturbations in the active site as compared to esterase activity has also been observed in other systems. It is for example a well-known fact that amidohydrolase activity of serine proteases can be easily damaged by site-directed mutagenesis [START_REF] Abrahamsén | Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution[END_REF] or chemical modifications [START_REF] Nakatsuka | Peptide segment coupling catalyzed by the semisynthetic enzyme thiolsubtilisin[END_REF][START_REF] West | Modification of protease to esterase for peptide synthesis: methyl chymotrypsin[END_REF][START_REF] Zhong | Active-site-directed modification of subtilisin[END_REF][START_REF] Plettner | Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants[END_REF][START_REF] Lloyd | Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity[END_REF]. In contrast, esterase activity was much less affected and even increased in some cases, although never to the extent reported herein for the Y312F mutant of FB188 HDAH. Thus, it appears that hydrolases, in general, require a more finely-tuned and stronger catalytic activity to hydrolyze energetically more demanding amides as compared to ester cleavage.

Implications for possible biological roles of histone deacetylase-like enzymes

and HDACs. In line with previous findings indicating that FB188 HDAH structurally and functionally resembles eukaryotic HDACs we observed that not only FB188 HDAH but particularly also recombinant human HDAC1 and HDAC3 exhibited pronounced esterase activity that far exceeded their amidohydrolase activity in terms of molar substrate conversion.

In regard of specificity, however, we noticed some differences. Whereas HDAC3/N-CoR2 and HDAC8 resembled FB188 HDAH in favoring the acetyl derivative of the ester substrate, HDAC1 showed a clear preference for the propionyl derivative. It can be speculated that esterase activity may have preceded amidohydrolase activity, which later in evolution may have been acquired through mutation of an active site residue (Tyr312 in HDAH). The mere existence of a pronounced esterase activity neither proves nor disproves that members of the histone deacetylase family at present have biological roles as esterases. The discovery of esterase activity, however, could be taken as an opportunity to reconsider our current view of histone deacetylases as specialized amidohydrolases and to motivate further research to identify biologically important esters as possible biological substrates of histone deacetylases.

Implications for HDAC inhibitor development.

It is a well-known fact that inhibitors of HDACs affect angiogenesis, cell-cycle arrest, apoptosis, terminal differentiation of different cell types and the pathogenesis of malignant disease [START_REF] Chung | Histone modification: the "next wave" in cancer therapeutics[END_REF]. The development of HDAC inhibitors has become a very promising therapeutic approach in modern cancer research [START_REF] Minucci | Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[END_REF]. However, mass screening for HDAC inhibitors, particularly in miniaturized formats, is still impeded by the relatively modest activities of the recombinant enzymes available. Furthermore, the most common fluorogenic assay in primary screening [START_REF] Wegener | A fluorogenic histone deacetylase assay well suited for high-throughput activity screening[END_REF] usually has to be performed as a two-step endpoint assay due to the susceptibility of most HDACs to trypsin [39]. In the light of our results it may be worthwhile to consider the esterase assay as a possible alternative [START_REF] Gronlund | Peptides: The Wave of the Future[END_REF]. In the esterase assay, substrate conversion rates are much more favorable for highly sensitive detection of small amounts of enzyme, particularly in miniaturized sample carriers.

Furthermore, the esterase-assay, e.g. with substrates C or D (Figure 1), permits continuous monitoring of enzyme activity. At least for all hydroxamate and trifluoromethylketone inhibitors tested so far the results of both assays, for example the IC50 values, are comparable. However, compound classes may exist for which the transferability of results is not guaranteed. Whatever assays will be used for HDAC inhibitor screening though, it appears mandatory to include major esterases into the inhibitor profiling. Our results indicate that at least certain hydroxamates and trifluoromethylketone inhibitors of HDACs are also very efficient lipase or esterase inhibitors and therefore could cause unwanted side effects in the treatment of patients.

FIGURE CAPTIONS

Fig. 1 .Fig. 2 .

 12 Fig. 1. Small molecule substrates of FB188 HDAH. (A) Boc-L-Lys(ε-acetyl)-MCA, (B) Boc-L-Lys(ε-propionyl)-MCA, (C) 4-methylcoumarin-7-acetate, (D) 4-methylcoumarin-7propionate, (E) 4-nitrophenyl-acetate.

Fig. 3 .Tab. 1 .

 31 Fig. 3. Structure of the active site of FB188 HDAH and proposed catalytic mechanism. (A) Crystallographic structure of inhibitor SAHA bound to the active site. (B) Schematic representation of interactions between SAHA and active site residues of FB188 HDAH. (C-E) Proposed mechanism for the deacetylation of amides (X = NH) and esters (X = O). HDAH residues are labelled.

Tab. 2 .

 2 IC50 values of HDAC inhibitors monitored by effects on amidohydrolase

Biochemical Journal Immediate Publication. Published on 12 Oct 2006 as manuscript BJ20061239 Copyright 2006 Biochemical Society

  named like in Figure1; P. fluorescens, Pseudomonas fluorescens; NA, no activity (background); nd, not determined

	4-methyl-coumarin-7-	0.90 ± 0.13	14100 ± 2100	1.68 ± 0.32 513000 ± 97700 1.66 ± 0.30	183 ± 41	2.84 ± 0.84	2150 ± 630
	acetate
	D 4-methyl-coumarin-7-	31.2 ± 3.7	2220 ± 260	0.22 ± 0.02	52400 ± 4700	2.54 ± 0.23	313 ± 28	nd	nd
	propionate
	E 4-nitrophenyl-acetate	11.8 ± 1.8	33000 ± 4900	nd	nd	47.0 ± 0.8	4850 ± 1220	nd	nd

*

Biochemical Journal Immediate Publication. Published on 12 Oct 2006 as manuscript BJ20061239 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 12 Oct 2006 as manuscript BJ20061239

ACKNOWLEDGEMENTS

This research was in part supported by grant BioFuture 0311852 from the Bundesministerium für Forschung und Technologie, Germany. Technical support from Cybio and BMG is greatly appreciated. HDAC1, HDAC3 and N-COR2 were a kind gift from H. Zhu, BPS Bioscience Inc. (San Diego, USA). The authors also would like to thank T. K. Nielsen for discussion and help in figure preparation. A.S. likes to thank G. Garrettson for critically reading the manuscript.


Tiago Braga

Mirjana Grujic

Agneta Lukinius

Lars Hellman

Magnus Åbrink

Gunnar Pejler
email: gunnar.pejler@bmc.uu.se

SERGLYCIN PROTEOGLYCAN IS REQUIRED FOR SECRETORY GRANULE INTEGRITY IN MUCOSAL MAST CELLS

Serglycin (SG) proteoglycans (PGs) are strongly implicated in the assembly of mast cell (MC) granules. However, this notion is mainly been based on studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In this study we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were in vitro differentiated into mucosal MC phenotype, expressing the markers mouse MC protease 1 (mMCP-1) and -2. Biosynthetic labelling experiments performed on these cells revealed an ~80% reduction of 35 SO 4 2-incorporation into PGs recovered from SG -/-cells as compared to SG +/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG +/+ and SG -/-cells but that their morphology was markedly affected by the absence of SG, e.g. with electron dense core formation only seen in SG +/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent on SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4, -6 and carboxypeptidase A were strongly dependent on SG for storage.

Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.

INTRODUCTION

Due to their location close to the host/environment surface, mast cells (MCs) 1) play a key role in the first line defence towards many invaders. For example, MCs have been shown to be important for combating infections caused by intestinal parasites [START_REF] Ha | Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice[END_REF][START_REF] Oku | Expulsion of Trichinella spiralis from the intestine of W/Wv mice reconstituted with haematopoietic and lymphopoietic cells and origin of mucosal mast cells[END_REF][START_REF] Nawa | Defective protective capacity of W/Wv mice against Strongyloides ratti infection and its reconstitution with bone marrow cells[END_REF] and bacteria [START_REF] Malaviya | Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha [see comments[END_REF][START_REF] Echtenacher | Critical protective role of mast cells in a model of acute septic peritonitis[END_REF]. In addition, it is now widely recognized that MCs contribute to adaptive immune responses in a variety of settings [START_REF] Galli | Mast cells in the development of adaptive immune responses[END_REF]. Although the specific phenotype of a MC population may vary to a large extent, dependent on exact tissue location and on exposure to stimuli, two major subclasses of MCs can be distinguished: mucosal MCs (MMCs) and connective tissue MCs (CTMCs) [START_REF] Metcalfe | Mast cells[END_REF].

CTMCs are, e.g., present in the skin, peritoneal cavity and intestinal submucosa, whereas MMCs are located in mucous membranes and in the intestinal lamina propria [START_REF] Metcalfe | Mast cells[END_REF]. Previous studies suggest that these two subtypes have different functions. For example, MMCs but not CTMCs increase in number when a parasite infection occurs [START_REF] Lutzelschwab | A kinetic analysis of the expression of mast cell protease mRNA in the intestines of Nippostrongylus brasiliensis-infected rats[END_REF][START_REF] Miller | Mucosal mast cells and the allergic response against nematode parasites[END_REF]. Moreover, MMCs are, in contrast to CTMCs, dependent on T-cells [START_REF] Mayrhofer | Mast cells in severely T-cell depleted rats and the response to infestation with Nippostrongylus brasiliensis[END_REF][START_REF] Ruitenberg | Absence of intestinal mast cell response in congenitally athymic mice during Trichinella spiralis infection[END_REF][12].

The different functional properties and tissue locations of CTMCs and MMCs are reflected by clearly distinct contents of secretory granule components, in particular as regards specific composition of proteases and proteoglycans (PGs) [START_REF] Welle | Development, significance, and heterogeneity of mast cells with particular regard to the mast cell-specific proteases chymase and tryptase[END_REF][START_REF] Miller | Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut[END_REF][START_REF] Caughey | New developments in the genetics and activation of mast cell proteases[END_REF]. While the CTMC subclass is known to contain MC carboxypeptidase A (CPA), the chymases mouse mast cell protease-4 (mMCP-4) and mMCP-5, as well as the tryptases mMCP-6 and mMCP-7 [START_REF] Reynolds | Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases[END_REF][START_REF] Serafin | Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker[END_REF][START_REF] Huang | Cloning and structural analysis of MMCP-1, MMCP-4 and MMCP-5, three mouse mast cell-specific serine proteases[END_REF], MMCs are instead characterized by a predominant expression of the chymases mMCP-1 and mMCP-2 [19,[START_REF] Serafin | Identification and molecular cloning of a novel mouse mucosal mast cell serine protease[END_REF].

Importantly however, the exact protease expression profile is dynamic, as shown by that MCs can alter their expression of proteases during the course of a parasite infection [START_REF] Friend | Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis[END_REF][START_REF] Friend | Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype[END_REF]. Moreover, in vitro exposure of bone marrow-derived MCs (BMMCs) to different cytokines will affect the specific profile of protease expression [START_REF] Haig | Effects of stem cell factor (kitligand) and interleukin-3 on the growth and serine proteinase expression of rat bonemarrow-derived or serosal mast cells[END_REF]24]. In addition to differential expression of protease genes, the different MC subclasses display distinct PG contents. Early studies provided evidence Copyright 2006 Biochemical Society that rat peritoneal MCs predominantly contained PGs of heparin type [START_REF] Yurt | Native heparin from rat peritoneal mast cells[END_REF], whereas intestinal MMCs instead contain predominantly CS of the "oversulphated" CS-E type (containing both 4-O-and 6-O-sulphated GalNAc units) [START_REF] Enerback | Glycosaminoglycans in rat mucosal mast cells[END_REF].

It has been assumed for as long time that the sulphated, and thereby negatively charged MC PGs mediate storage of various basically charged secretory granule components [START_REF] Metcalfe | Mast cells[END_REF]. Strong support for this notion came when two groups showed simultaneously that interference with the heparin biosynthesis in CTMCs, by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2), caused dramatic defects in their storage of secretory granule proteases [27,[START_REF] Humphries | Heparin is essential for the storage of specific granule proteases in mast cells [see comments[END_REF]. In contrast, BMMCs were affected only to a limited extent by the NDST-2 knockout, most likely due to their preferential synthesis of CS rather than heparin [START_REF] Humphries | Heparin is essential for the storage of specific granule proteases in mast cells [see comments[END_REF][START_REF] Henningsson | Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing[END_REF]. Noteworthy, however, is that the small amounts of heparin present in BMMCs appear to be essential for the processing of CPA and storage of mMCP-5, although granular morphology and mMCP-6 storage is not affected [START_REF] Humphries | Heparin is essential for the storage of specific granule proteases in mast cells [see comments[END_REF][START_REF] Henningsson | Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing[END_REF].

Moreover, because the MMCs do not contain heparin they were not affected by the NDST-2 knockout, and the possible role of PGs in secretory granule homeostasis in MMCs has therefore remained an open question.

In the PGs of both CTMCs and MMCs it has been believed that the respective glycosaminoglycan (GAG) chains (CS or heparin) are attached to the same protein "core", namely serglycin (SG). In a recent study we targeted the SG gene and found, indeed, that CTMC granules showed similar, although more profound, defects as those previously observed after interference with NDST-2 [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. In addition, we found that bone marrow cells differentiated into a CTMC-like phenotype were affected to a similar extent as in vivo-derived CTMCs [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. Here we used the SG -/-mouse strain to specifically address the role of SG PG in MMCs. We show that SG PG is the dominating cell-associated PG in MMCs and that it is needed for assembly of mature EXPERIMENTAL MMCs. In vitro-derived MMC-like cells were obtained by culturing femur and tibia bone marrow cells from SG +/+ and SG -/-littermates as described previously [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. For the majority of experiments, mice backcrossed to the C57BL/6 genetic background were used. For analysis of mMCP-7, mice were backcrossed for 10 generations to DBA-1 background. The cell cultures were maintained at a concentration of ~ 0.5 x 10 6 cells/mL in medium supplemented with recombinant human TGF-β (1 ng/mL; Peprotech, London, UK), mouse IL-3 (1 ng/mL; Peprotech), mouse IL-9 (5ng/mL; Peprotech) and mouse stem cell factor (SCF; 50 ng/mL; Peprotech), according to a previously described protocol [START_REF] Miller | A novel function for transforming growth factor-beta1: upregulation of the expression and the IgEindependent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1[END_REF]. Medium and cytokines were changed every 2-3 days, for 14 days.

Staining procedures. Cells were collected onto cytospin slides and were stained with May Grünwald/Giemsa [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. The chloroacetate assay was performed on cytospin slides as follows: 0.5 g of pararosaniline base (Sigma) were dissolved in 20 mL H 2 O and 2.5 mL of HCl during heating, followed by filtering. To 0.1 mL of this solution was then added 0.1 mL of 4% sodium nitrite (Merck) to yield nitrosylated pararosanilin. Solution A (2.73 g Na 2 HPO 4 in 250 mL H 2 O) and solution B (2.27 g KH 2 PO 4 in 250 mL H 2 O) were prepared and mixed (41 mL A and 9 mL B) to prepare solution C. Cytospin slides were placed in a solution containing 10 mg Naphtol-ASD chloroacetate (Sigma), 1 mL N,N-dimethylformamide (Sigma), 35 mL solution C and 0.2 mL nitrosylated pararosanilin solution (contents were combined in the order given, followed by filtering) for 40 min, after which they were washed in H 2 O for 5 minutes and air dried. Copyright 2006 Biochemical Society Small intestines were excised and fixed in either 4% paraformaldehyde or Carnoy's fixative followed by embedding in paraffin, sectioning and staining with acidic toluidine blue.

Extraction of intestinal protein. Small intestines were excised from SG +/+ and SG -/-mice, washed with PBS and stored at -70°C. After defrosting, tissues were first homogenized (using a PT1200 Polytron device; Kinematica AG; Lucerne, Switzerland) in 1.5 mL of low salt PBS buffer (pH 7.4; 0.14 M NaCl) containing 1 mM EDTA, 1% Triton X-100, 0.14 M NaCl and a protease inhibitor cocktail (final concentrations: 2.3 mM AEBSF, 10 mM EDTA, 0.2 mM Bestatin, 0.03 mM pepstatin A, 0.03 mM E-64) (Sigma-Aldrich), followed by centrifugation (13,400 rpm, 4°C, 20 min). Supernatants were collected and pellets were homogenized in a high salt PBS buffer (pH 7.4; 2 M NaCl) containing 1 mM EDTA, 1% Triton X-100, 2 M NaCl and protease inhibitor cocktail as above. Samples were rotated for 30 min. at 4°C, centrifuged (13,400 rpm, 4°C, 20 min) and the resulting supernatants were collected.

Immunoblot analysis. Extracts from small intestines or bone marrow-derived MMCs were analyzed for the presence of mMCP-1, -2, -4, -5, -6 and CPA by utilizing specific rabbit antisera, according to the methods described previously [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. Immunoblot analysis for mMCP-7 was carried out with a rat antiserum towards recombinant mMCP-7.

Zymography. Samples from MMC-conditioned media or cell extracts were analyzed by gelatin zymography as described elsewhere [START_REF] Fajardo | Human mast cell beta-tryptase is a gelatinase[END_REF]. Matrix metalloprotease 2 (MMP-2) and MMP-9 were identified by comparison of their migration velocities with those of standard human MMP-2 and -9 present in conditioned medium from the HT-1080 cell line. Copyright 2006 Biochemical Society GAG isolation and analysis. Bone marrow-derived MMCs (20 x 10 6 cells) were biosynthetically labelled overnight with 0.32 mCi of carrier-free 35 S-sulphate (Amersham Biosciences). Cells were pelleted by centrifugation, followed by the purification of cell-associated and secreted PGs as described previously [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. Samples (10,000 cpm) of isolated 35 S-labelled GAGs were mixed with 0.5 mg of CS-A (containing 4-O-sulphated GalNAc residues; Sigma) and 0.45 mg of pig mucosal heparin (gift from Ulf Lindahl, Uppsala University, Sweden) and were analyzed by anion exchange chromatography on DEAE-Sephacel [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. To release GAG chains from intact PGs, samples of purified PGs were treated with 0.5 M NaOH over night, followed by neutralization with HCl and subsequent analysis on a Superose 6 column (Amersham Biosciences, Uppsala, Sweden) eluted with 0.2 M NH 4 HCO 3 at 0.25 ml/min. Fractions (0.5 ml) were collected and were analyzed for 35 S-radioactivity by scintillation counting.

To determine the CS content, samples of labelled GAGs were treated with 0.05 units of chondroitinase ABC (Seikagaku, Tokyo, Japan) in 20 µL of 0.05 M Tris-HCl (pH 8.0)/0.3 M NaCl/1 mg/mL bovine serum albumin, and incubated at 37°C for 15 h. Samples were analyzed by gel chromatography on a Sephadex G-50 column (0.5 x 90 cm) (Amersham Biosciences), eluted with 0.2 M NH 4 HCO 3 . 0.5 mL fractions were collected and analyzed for 35 S-radioactivity.

For papain digestion, 5000 cpm were evaporated to dryness and resuspended in 300 uL of 50 mM sodium acetate (pH 5.5)/ 2M NaCl. To each sample, 25 uL 0.5M cysteinium chloride, 25uL 0,5M EDTA and 50 U papain (Sigma) were added. Samples were incubated for 18h, at 56°C with shaking. Reactions were stopped by heating for 5 min. at 100°C, followed by centrifugation (5 min, 600 g).

RT-PCR analysis. Total RNA was isolated using Total RNA Isolation -Nucleospin RNA II kit (Machery-Nagel) using the protocol provided by the manufacturer. For RT-PCR, Superscript II Copyright 2006 Biochemical Society (Invitrogen) and specific reverse primers were used in order to produce single-stranded cDNA.

Hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as a house keeping control.

The primers used, except the primers for mMCP-7, were as described previously [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. The primers for mMCP-7 were: 5'-TGC CAA TGA CAC CTA CTG GAT GC-3' (forward) and 5'-CAG GAG TCA TGT CCT TCA TTC C-3' (reverse; yielding a 512 bp product). RT-PCR was performed using the following steps: 95°C (2 min), 95°C (30 s), 60°C (30 s), 72°C (1 min), 35 times to step 2, 72°C (10 min). PCR products were sequenced to confirm their identity with the proposed target sequence.

Transmission electron microscopy (TEM). Cells were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, supplemented with 0.1 M sucrose for 10 h. Next, cells were post-fixed in 1% osmium tetroxide in the same buffer for 90 min., dehydrated in graded series of ethanol and embedded with the epoxy plastic Agar 100 (Agar Aids, Stansted, UK). Ultrathin sections were placed on Formvar-coated copper grids and constrated with 2% uranyl acetate and Reynolds Lead citrate. Analysis was performed in a Hitachi Electron Microscope at 75 kV.

RESULTS

To study the role of SG PG in MMCs we recovered bone marrow cells from SG +/+ and SG -/- animals and cultured them in medium containing TGF-β, stem cell factor (SCF), IL-3 and IL-9, conditions that were previously shown to induce a homogenous MMC-like phenotype as regards protease content [START_REF] Miller | A novel function for transforming growth factor-beta1: upregulation of the expression and the IgEindependent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1[END_REF]. After 2 weeks of culture, SG +/+ and SG -/-cells were recovered and their morphological and biochemical characteristics were compared. As shown in Fig. 1A (left panels), SG +/+ in vitro-derived MMC-like cells showed strong metachromatic granular staining with May Grünwald/Giemsa, a typical feature of mature MCs. In contrast, SG -/-cells were not stained.

Instead, May Grünwald/Giemsa-negative, "empty"-appearing vesicles were clearly visible (Fig.

1A: right panel); such vesicles were also visible, although to a lesser extent, in SG +/+ MCs. As is evident from Fig. 1A (upper panels), cells of SG +/+ and SG -/-genotype were similar in terms of shape and size, indicating that the cell culture conditions gave rise to homogenous cell populations. In order to address the in vivo relevance of these findings, tissue sections of small intestines were stained with toluidine blue. As shown in Fig. 1B, rare metachromatically stained cells were observed both in the mucosal (left panel) and in the submucosal (right panel) region of the small intestine from SG +/+ mice, indicating the presence of MMCs and CTMCs, respectively.

In contrast, toluidine blue-positive cells were undetectable both in the intestinal mucosa and submucosa of SG -/-animals. These results indicate that the absence of SG PG causes dramatic defects in staining of MMCs with cationic dyes, both in vivo and in in vitro-derived MMC-like cells.

To analyze the importance of SG PGs for secretory granule organization in more detail, SG +/+ and SG -/-cells were analyzed by TEM. TEM analysis of both SG +/+ and SG -/-cells revealed Copyright 2006 Biochemical Society normal ultrastructure of membranes, mitochondria and other features, indicating optimal culturing conditions and preparation procedure. Proliferating cells were frequently seen in both cell cultures. Granules in SG +/+ cells were spherical and displayed diversity in the arrangement of substructures in the interior of each granule. Three main types of granules could be distinguished in SG +/+ cells: i) granules containing small vesicles, most likely exosomes [START_REF] Skokos | Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo[END_REF], that were filled with electron dense material, ii) granules containing small vesicles together with a grainy substance of moderate to high electron density, or iii) granules containing grainy substance of high electron density, without other substructural elements (Fig. 2 A-C). Generally, SG +/+ granules were divided into distinct compartments of low electron density (electron translucent) and electron dense cores (Fig. 2 A-C). Notably, the granule types identified in the in vitro-derived MMCs resemble those previously identified in BMMCs developed in the presence of conditioned medium from Con A-stimulated splenocytes [START_REF] Raposo | Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation[END_REF]. The granule morphology of SG -/-cells was markedly different. Firstly, granules of SG -/-cells were slightly smaller than those in SG +/+ cells and often had an ovoid shape. Furthermore, most of the SG -/-granules lacked the intragranular vesicles (exosomes) that were often present in SG +/+ counterparts, although solitary granules containing such vesicles could be observed. Another striking difference between SG +/+ and SG -/- granules was that the granules of SG -/-cells lacked the subdivision into electron dense-and translucent regions that was typical for the SG +/+ granules. Instead, SG -/-granules were usually completely filled with material of intermediate to relatively high electron density. Moreover, the contents of the SG -/-granules were of a more amorphous character and lacked the typical formation of grainy electron dense core that was seen in SG +/+ granules (Fig. 2 D-F). These results are clearly consistent with a role for SG in the granule maturation process, with the amorphous appearance of the SG -/-granules resembling the immature granules seen in other , and labelled GAGs were subsequently recovered both from the conditioned media (secreted GAGs) and from the cell layer. Quantification of the total amount of radioactivity in each pool showed that the cell layer of SG +/+ cells contained ~5-fold higher amounts of labelled material than the corresponding pool recovered from SG -/-cells (Table 1), indicating that SG is the main cell-associated PG in in vitro-derived MMC-like cells. In contrast, similar amounts of labelled GAGs were recovered in conditioned media from SG +/+ and SG -/-cells, indicating that SG is not secreted to a major extent by the in vitro-derived MMC-like cells. (Table 1).

To further characterize the nature of the GAGs, samples of 35 S-labelled material were subjected to anion exchange chromatography along with internal standards of CS-A (low degree of sulfation; ~1 sulphate groups/disaccharide) and heparin (highly sulphated; ~2.5 sulphate groups/disaccharide unit). As shown in Fig 3A andD, respectively, most of the cell-associated and secreted material from SG +/+ cells showed co-elution with standard CS-A, indicating a relatively low degree of sulfation. However, a small shoulder of material eluted slightly later in the salt gradient. Cell-associated (Fig. 3B) and secreted (not shown) material from SG -/-cells displayed a similar pattern of charge density as the corresponding samples from SG +/+ counterparts, indicating that 35 S-labelled macromolecules of SG-and non-SG species have Copyright 2006 Biochemical Society similar anionic charge densities. For a comparison, 35 S-labelled material from SG +/+ and SG -/- bone marrow cells that had been in vitro differentiated into a more CTMC-like phenotype [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF] were analyzed. Interestingly, GAGs derived from SG +/+ CTMC-like cells eluted markedly later in the salt gradient as compared with the corresponding material derived from in vitro-derived MMC-like cells, indicating a substantially higher charge density (Fig. 3C). Further, GAGs from SG -/-CTMC-like cells were eluted clearly before the corresponding material recovered from SG +/+ counterparts (Fig. 3C). Together, these results indicate that SG PGs in CTMC-like cells have markedly higher charge densities than non-SG PGs, whereas SG-and non-SG species in in vitro-derived MMC-like cells display similar anionic charge. Further, these findings introduce the possibility that the cytokine mixture that preferentially induces a MMC-like phenotype directs the GAG synthesis, by unknown mechanisms, towards the assembly of lower sulphated species as compared to CTMC GAGs. Since SG PG can accommodate GAGs of either heparin-or CS type as side chains it was important to investigate which class of GAG that was attached to the SG core protein. Chondroitinase ABC digestion resulted in depolymerization of most of the labelled GAGs from SG +/+ cells (Fig. 3E). In addition, most of the GAGs from the SG -/-cells were depolymerised by chondroitinase ABC. Hence, most of the GAGs attached both to SG-and non-SG species of PGs in in vitro-derived MMC-like cells are of CS type.

To obtain further information on the macromolecular properties of the PGs, the purified 35 Slabelled macromolecules were analyzed by size exclusion chromatography on a Superose 6 column. In addition, material was analyzed after treatment with 0.5 M NaOH to release GAG chains from its protein core. As shown in Fig. 3F, most of the labelled material from SG +/+ cells eluted as a single peak. Radiolabelled material from SG -/-cells eluted somewhat later, indicating a lower molecular size. Alkali treatment of 35 S-labelled macromolecules from the SG +/+ in vitroderived MMC-like cells caused a marked right shift of the peak, indicating release of GAG Copyright 2006 Biochemical Society chains bound to the core protein (Fig. 3F). In contrast, alkali treatment of labelled material from SG -/-cells caused only a minor a right shift of the peak, indicating that the 35 S-labelled GAGs recovered from SG -/-cells were mainly in the form of free GAG chains. Noteworthy, the GAG chains derived from SG -/-cells eluted before the corresponding GAGs present in SG +/+ cells, indicating that GAGs attached to non-SG species have a higher average molecular weight than those connected to the SG protein core.

A typical feature of SG PGs is their resistance to protease treatment, explained by their small protein core that is inaccessible to proteolytic cleavage due to dense substitution with GAG chains [START_REF] Yurt | Native heparin from rat peritoneal mast cells[END_REF][START_REF] Zernichow | Serglycin is the major secreted proteoglycan in macrophages and has a role in the regulation of macrophage tumor necrosis factor-alpha secretion in response to lipopolysaccharide[END_REF]. In order to address whether this is a characteristic of SG PGs from in vitroderived MMC-like cells, radiolabelled PGs were treated with papain, followed by gel filtration analysis. Indeed, PGs from SG +/+ cells were to a large extent (~70%) resistant to treatment.

Papain treatment of 35 S-labelled macromolecules from SG -/-cells caused a slight right shift, similar to that caused by alkali treatment, consistent with the notion that GAGs from SG -/-cells were mainly present in the form of free polysaccharide chains (results not shown).

MC granules are typically characterized by strong chloroacetate esterase activity, attributable to their high content of chymase activity [START_REF] Huntley | Histochemical demonstration of chymotrypsin like serine esterases in mucosal mast cells in four species including man[END_REF]. In a previous report we showed that the chloroacetate esterase activity in CTMCs was strongly dependent on the presence of SG PG [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. Experiments were therefore carried out to investigate if also MMC chloroacetate esterase activity was dependent on the presence of SG PG. However, in striking contrast to CTMCs, chloroacetate esterase activities were similar in SG +/+ and SG -/-in vitro-derived MMC-like cells (Fig. 1C).

Thus, the protease(s) responsible for the chloroacetate esterase activity in in vitro-derived MMClike cells does not appear to depend on SG. This further implies that the MMC proteases may display a different degree of SG-dependence as compared to the CTMC proteases. Experiments Copyright 2006 Biochemical Society were therefore performed to compare the protease profiles of SG +/+ and SG -/-MMCs, both at the mRNA and protein levels. RT-PCR analysis showed that the in vitro-derived MMC-like cells displayed clearly detectable transcripts for the MMC markers mMCP-1 and mMCP-2, indicating that the in vitro culture conditions employed indeed gave cells of MMC-like phenotype. Further, transcripts were detected for mMCP-4 and mMCP-5, in agreement with a previous report [START_REF] Miller | A novel function for transforming growth factor-beta1: upregulation of the expression and the IgEindependent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1[END_REF], and for mMCP-6 and CPA (Fig. 4A). Importantly, all of these protease genes were expressed both in SG +/+ and SG -/-cells, indicating that the lack of SG did not affect the degree or direction of cellular differentiation (Fig. 4A).

Next, experiments were carried out to study how the lack of SG affected the proteases at the protein level. Immunoblot analysis of cell extracts from in vitro-derived MMC-like cells revealed the presence of mMCP-1 antigen in cells of SG +/+ genotype. Interestingly, mMCP-1 protein was present in similar amounts in SG -/-counterparts, indicating that this protease is not dependent on SG PG for storage (Fig. 4B). Also mMCP-2 protein was detected in SG +/+ cells but in contrast to mMCP-1, mMCP-2 levels were somewhat reduced in SG -/-cells, indicating a partial dependence on SG for storage. A much more pronounced dependence on SG PG for storage was seen for mMCP-4, mMCP-6 and CPA, with antigen levels being close to undetectable in cells of SG -/- genotype (Fig. 4B). mMCP-5 protein was detected in both SG +/+ and SG -/-in vitro-derived MMClike cells, but with a marked decrease of mMCP-5 levels in the absence of SG (Fig. 4B).

To test if these results could be confirmed in vivo, small intestines from SG +/+ and SG -/-mice were homogenized by a two step procedure, first with a low salt-and then with a high salt buffer.

Immunoblot analysis of the corresponding extracts reproduced the patterns seen in the in vitro-

derived MMC-like cells, i.e. mMCP-1 antigen was present in similar amounts in SG +/+ and SG -/- tissue, mMCP-2 showed a partial reduction in SG -/-tissue, whereas mMCP-6 and CPA were Copyright 2006 Biochemical Society essentially undetectable in SG -/-small intestine. Interestingly, the different MC proteases were recovered in different extraction pools. mMCP-1 and -2 were both recovered exclusively in the low salt extract, indicating a low degree of electrostatic interaction with other tissue components (Fig. 5). In contrast, a large part of the total mMCP-6 and CPA antigen required high salt conditions for extraction, indicating tight electrostatic interactions with anionic components of the tissue (Fig. 5).

The experiments described above were all performed on mice of C57BL/6 background. This mouse strain expresses mMCP-1, -2, -4, -5, -6 and CPA but lacks the tryptase mMCP-7 due to the presence of a premature stop codon in the mMCP-7 gene [START_REF] Hunt | Natural disruption of the mouse mast cell protease 7 gene in the C57BL/6 mouse[END_REF]. Thus, it is not possible to investigate the dependence of mMCP-7 on SG using the C57BL/6 strain. In order to address this issue we therefore backcrossed the SG -/-strain to DBA-1 genetic background for 10 generations, and then analyzed in vitro-derived MMC-like cells from this strain as well as ear skin tissue for mMCP-7 expression and dependence on SG for storage. As shown in Fig. 6A, in vitro-derived MMC-like cells from SG +/+ and SG -/-mice of DBA-1 background showed expression of the mMCP-7 gene as determined by RT-PCR analysis. Moreover, mMCP-7 antigen was clearly detected in cell extracts from SG +/+ in vitro-derived MMC-like cells from DBA-1 mice as determined by immunoblot analysis, but as expected not in extracts obtained from corresponding cells of C57BL/6 background (Fig. 6B). mMCP-7 protein was also detected in extracts from SG -/- cells of DBA-1 background, with the level of mMCP-7 being similar as in extracts from SG +/+ cells (Fig. 6B). This indicates that mMCP-7 storage does not depend on SG. mMCP-7 antigen was also detected in extracts from ear skin tissue, again with the level of mMCP-7 being similar in extracts from SG +/+ and SG -/-mice. The latter indicates that mMCP-7 storage is independent on SG also at the in vivo level. For a comparison, SG +/+ and SG -/-in vitro-derived MMC-like cells from mice of DBA-1 background were also analyzed for mMCP-6 antigen. In accordance with Copyright 2006 Biochemical Society results performed on cells from mice of C57BL/6 background, mMCP-6 antigen levels were markedly reduced in SG -/-cells (not shown).

In addition to the MC-specific proteases (see above), MCs are known to express MMPs [START_REF] Fang | Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase[END_REF].

Since the potential dependence of the MMPs on PGs for storage or secretion has not been investigated previously, we chose to address this issue. Conditioned media as well as cell extracts were collected from SG +/+ and SG -/-in vitro-derived MMC-like cells (of C57BL/6 background), and were analyzed by gelatin zymography. Both MMP-2 and MMP-9 were clearly detected in conditioned medium from SG +/+ and SG -/-cells. However, the levels were similar in medium obtained from SG +/+ and SG -/-cells, indicating that the expression/secretion of MMP-2 and MMP-9 is not dependent on SG (results not shown). No detectable MMP activity was seen in the corresponding cell extracts, indicating that MMP-2 and -9 are not stored to any large extent in in vitro-derived MMC-like cells (not shown). However, it is important to stress that bone marrow-derived CTMC-like cells differ in many aspects from the CTMCs found in vivo. For example, in vitro-derived CTMC-like cells synthesize mainly GAGs of CS type, whereas in vivo-derived CTMCs produce predominantly heparin [START_REF] Yurt | Native heparin from rat peritoneal mast cells[END_REF]27]. Strikingly though, the GAGs recovered both from in vivo-derived CTMCs and from in vitro-derived counterparts display conspicuously high charge density [START_REF] Yurt | Native heparin from rat peritoneal mast cells[END_REF][START_REF] Henningsson | Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing[END_REF]. Thus, although the major GAGs of in vivo-and in vitro-derived CTMCs are of different species, their degree of sulfation is conserved.

In contrast to the CTMC PGs, relatively little has been known concerning the characteristics and function of the PGs present in MMCs. In an early report it was shown that intestinal MMCs of rats infected with Nippostrongylus brasiliensis synthesized large amounts of oversulphated CS [START_REF] Enerback | Glycosaminoglycans in rat mucosal mast cells[END_REF]. However, the nature of the MMC PG, i.e. which protein core to which the CS chains are attached to, and the role of the MMC PGs in granule homeostasis has previously not been Here we show that MMCs with an inactive SG gene show an ~80% reduction in the amount of 35 S-labelled PGs recovered from the cell layer, strongly indicating that SG is the major cell-associated PG in in vitro-derived MMC-like cells. In CTMC-like BMMCs, the lack of SG caused a similar reduction of cell-associated PGs [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF], thus suggesting that SG accounts for most of the cellular PGs in both of these MC classes. However, it is important to stress that the lack of SG did not result in a complete lack of cell-associated PGs either in MMC-or CTMC-like BMMCs, indicating that PGs of other species than SG are expressed. To date, the possible expression and cellular location of PG species other than SG in MCs has not been well studied.

However, considering that the absence of SG resulted in an essentially complete lack of granular staining with cationic dyes, it is likely that SG accounts for most of the secretory granule PGs. Therefore, we may suggest that non-SG PGs expressed by MCs are mostly cell-surface associated species. In contrast to the effect on cell-associated PGs, the absence of SG did not influence the level of secreted PGs. The most probable explanation for this observation is that the PGs secreted by the in vitro-derived MMC-like cells are of non-SG type.

We show here that bone marrow cells cultured with TGF-β/SCF/IL-3/IL-9 synthesize PGs of relatively low charge density. This is in sharp contrast to the PGs synthesized by bone marrow cells that have been driven into a more CTMC-like phenotype. Although both populations of BMMCs synthesize predominantly SG PGs with attached CS chains, the latter cell population synthesizes PGs of markedly higher anionic charge density. This indicates that the level of sulfation of a SG PG is highly dependent on the specific cytokine environment. Possibly, the regulation of sulfation density is achieved by altering the levels or activities of the sulfotransferases responsible for introducing the 4-O-and 6-O-sulphate groups into GalNAc residues in CS. However, the mechanisms involved in the regulation of these enzymatic activities Copyright 2006 Biochemical Society [START_REF] Hunt | Natural disruption of the mouse mast cell protease 7 gene in the C57BL/6 mouse[END_REF]. We show that, in contrast to mMCP-4, -5, -6 and CPA, mMCP-1 and mMCP-7 are independent on SG for storage, whereas mMCP-2 is only partially dependent on SG. The most likely explanation for these different degrees of dependence on SG for storage lies within the cationic properties of the respective proteases. Most of the CTMC proteases (mMCP-4, -5 and CPA) display high net positive charges (Table 2) and it has been shown by molecular modelling that the basic residues of mMCP-4 and -5 are contained within two separate and defined patches [START_REF] Sali | Threedimensional models of four mouse mast cell chymases. Identification of proteoglycan binding regions and protease-specific antigenic epitopes[END_REF]. Possibly, this organization of basic charge may promote multivalent and thereby strong interactions with anionic PGs. In contrast, mMCP-1 has a markedly lower net charge (Table 2) and, moreover, mMCP-1 lacks one of the two basic clusters present in mMCP-4 and -5. The partial dependence of mMCP-2 on SG for storage is intriguing. Interestingly however, the net positive charge of mMCP-2 is intermediate between those of mMCP-1 versus mMCP-4/mMCP-5/CPA (Table 2), thus providing a likely explanation for its intermediate dependence on SG for storage. In a recent report we studied the SG-dependence of various components of the lytic granules of cytotoxic T lymphocytes for storage [START_REF] Grujic | Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage[END_REF]. Also in that study we found a distinct correlation between exposure of positively charged patches on the molecular surface and dependence on SG for storage. We showed that granzyme B was strongly dependent on SG for storage, and that this may depend on a defined basic patch on the surface of the molecule. In contrast, granzyme A was independent on SG for storage despite its high net positive charge (Table 2), possibly due to a less defined basic region on the molecular surface [START_REF] Grujic | Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage[END_REF].

At first view, the strong dependence of mMCP-6 on SG for storage may seem contradictory, considering its net negative charge (Table 2). However, mMCP-6 displays a contiguous cluster of His residues at the molecular surface and, at the acidic pH prevalent in secretory granules, these His residues are positively charged and engage in tight electrostatic interactions with GAGs [START_REF] Hallgren | Histidines are critical for heparin-dependent activation of mast cell tryptase[END_REF].

Interestingly, we show here that the second major secretory tryptase in murine MCs, mMCP-7, Copyright 2006 Biochemical Society appears to be independent on SG PGs for storage. In line with such a differential dependence of mMCP-6 and -7 on SG PG, it has been shown that mMCP-6 is preferentially retained at the surface of degranulated MCs following passive anaphylaxis, possibly through a firm association with SG PG even after exocytosis, whereas mMCP-7 readily diffuses away from the degranulated MCs into the circulation [START_REF] Ghildyal | Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues, and rapid accumulation of enzymatically active mMCP-7 in the blood[END_REF]. In further support for a weaker interaction of mMCP-7 than mMCP-6 with SG PG, the His cluster that was shown to mediate interaction between mMCP-6 and heparin is not fully conserved in mMCP-7 [START_REF] Hallgren | Histidines are critical for heparin-dependent activation of mast cell tryptase[END_REF]. Moreover, a Lys/Arg patch that has been identified on the surface of mMCP-6 by molecular modelling, and has been suggested to engage in the interactions with GAGs, is absent in mMCP-7 [START_REF] Ghildyal | Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues, and rapid accumulation of enzymatically active mMCP-7 in the blood[END_REF].

Based on the findings presented here and on those presented previously [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF][START_REF] Grujic | Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage[END_REF] we may suggest a general model for the role of SG PG in the organization of secretory granule. As is evident from this work, the actual secretory vesicles are assembled independently of SG PG. Moreover, transport of secretory compounds into the granules does not appear to depend on SG PG.

However, the maturation of the secretory granule with electron dense core formation is dependent on SG PG. Certain granular constituents, e.g. mMCP-4, -6, CPA and granzyme B are highly dependent on SG for storage [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF][START_REF] Grujic | Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage[END_REF], indicating their presence within the electron dense cores. In contrast, the lack of SG dependence seen for granzyme A, mMCP-1 and mMCP-7 suggests that these compounds are not included within the electron dense cores. It is reasonable to assume that compounds not interacting with SG are not retained in the granule to the same extent as the components that are tightly associated with SG. Indeed, it has been shown that mMCP-1 is constitutively secreted from MMCs, clearly consistent with such a notion [START_REF] Wastling | Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection[END_REF][START_REF] Brown | Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues[END_REF]. Taken together, SG may thus regulate the composition of secretory granules by binding to certain compounds whereas others are independent on SG for storage. The fate of the SG-dependent proteases in cells lacking SG is an intriguing issue. Although this issue is still not completely Copyright 2006 Biochemical Society

  secretory granule-containing cells, e.g. immature β-cells of the pancreas [35]. Further, the Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257 Copyright 2006 Biochemical Society subdivision of SG +/+ granules into regions of high and low electron densities is clearly in line with the granule morphology seen in more mature pancreatic β-cells [35]. Importantly, the lack of SG PG did not affect the cellular morphology apart from its effects on secretory granules. Next, experiments were undertaken to characterize the GAGs synthesized by the in vitro-derived MMC-like cells and to study the relative contribution of GAGs present in PGs of SG type to the total synthesis of GAGs. In vitro-derived MMC-like cells were biosynthetically labelled with 35

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

  of MCs is their strong metachromatic staining with cationic dyes. These staining properties are dependent on the large amounts of negatively charged GAGs that are present in the MC secretory granules. However, different MC subclasses express different GAG species, i.e. heparin or CS. Moreover, the degree of sulfation of the respective GAG may vary significantly depending on MC subclass and tissue location. Although this has been recognized for a long time, very little is known regarding the factors that regulate this diversity. In this study we have specifically addressed the PGs of MMCs. Detailed studies of this MC subclass have recently been made possible through the identification of cytokine mixtures that specifically promote the differentiation of bone marrow precursors into MCs of MMC-like phenotype[START_REF] Miller | A novel function for transforming growth factor-beta1: upregulation of the expression and the IgEindependent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1[END_REF], whereas previous protocols have mainly resulted in MCs of more CTMC-like phenotype[24].

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Fig. 1 .

 1 Fig. 1. Morphology and phenotype of mucosal type bone marrow-derived MCs. (A)Cytospin slides were prepared from bone marrow cells recovered from SG +/+ and SG -/-mice after

Fig. 2 .Fig. 4 .

 24 Fig.2. Transmission electron microscopy. (A) Representative micrograph from SG +/+ in vitro-

Fig. 5 .

 5 Fig. 5. Immunoblot analysis of MC proteases in intestinal extracts. Intestinal extracts were prepared from SG +/+ and SG -/-mice by a two-step procedure, first with a low salt (LS) buffer and

Fig. 6 .Fig. 3 Biochemical

 63 Fig.[START_REF] Galli | Mast cells in the development of adaptive immune responses[END_REF]. mRNA and immunoblot analysis of mMCP-7. (A) In vitro-derived MMC-like cells were cultured from SG +/+ and SG -/-mice of DBA-1 genetic background. After 4 days of culture,

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257 Copyright 2006 Biochemical SocietyTable 1 .

 1 Incorporation of 35 SO 4 2-into PGs from in vitro-derived MMC-like cells. Results are expressed as cpm incorporated/1 x 10 6 cells. Results are representative of three independent experiments.

	Source of 35 S-labelled PGs	SG +/+	SG -/-
	Secreted PGs	10,000	9,000
	Cell-associated PGs	107,000	22,000

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257 Copyright 2006 Biochemical SocietyTable 2 .

 2 Net charge of selected proteases and their dependence on SG for storage.

		Net charge 1) SG-dependence
	Protease	(pH 7.0)	for storage	Reference
	mMCP-1	+4.7	-	This study
	mMCP-2	+7.2	(+)	This study
	mMCP-4	+16.7	+	This study and [30]
	mMCP-5	+13.9	+	This study and [30]
	mMCP-6	-2.2	+	This study and [30]
	mMCP-7	-6.2	-	This study
	CPA	+18.5	+	This study and [30]
	Granzyme A	+15.3	-	[41]
	Granzyme B	+18.9	+	[41]

1)

Net charges were calculated for mature proteins, i.e. without activation peptide.

Biochemical Journal

Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061257

ACKNOWLEDGEMENTS This research project has been supported by a Marie Curie Early Stage Research Training

Fellowship of the European Community's Sixth Framework Programme under contract number 504926, the Swedish Research Council, King Gustaf V's 80th anniversary Fund and the Mizutani Foundation for Glycoscience.

FOOTNOTES

in MCs remain to be studied. Nevertheless, the distinct anionic charge densities of the PGs, depending on whether the MCs have been differentiated into MMC-or CTMC-like phenotype strongly support that the different culture conditions resulted in distinct MC subtypes.

Considering the major effects on granule morphology seen in CTMCs following the knockout of either NDST-2 or SG, it may be speculated that SG PG may actually participate in the formation of secretory vesicles. However, the ultrastructural studies presented here clearly show that granules are formed irrespectively of the presence or absence of SG. Importantly though, the granule morphology differed markedly in SG +/+ and SG -/-in vitro-derived MMC-like cells. In SG +/+ cells, granules contained distinct areas of low-and high electron density, with the electron dense material assembled into a grainy and condensed dense core. In contrast, SG -/-granules lacked this substructural division, with amorphous material occupying the entire granule space.

We may therefore suggest that SG PG is not necessary for the intracellular transport of compounds into the granule but that SG is needed for the condensation of granular constituents.

Most likely, the mechanism of condensation involves electrostatic interactions between basically charged domains of the respective granule compounds and the anionic GAGs attached to the SG protein core.

During the studies of CTMCs it was found that all of the characteristic CTMC secretory granule proteases expressed in the C57BL/6 strain, i.e. mMCP-4, -5, -6 and CPA, were strongly dependent on SG PG for storage [START_REF] Abrink | Serglycin is essential for maturation of mast cell secretory granule[END_REF]. Importantly, the possible dependence of the MMC proteases on SG for storage has not been investigated previously, and we therefore addressed this issue here. Further, the possible dependence of mMCP-7 on SG for storage has not been investigated previously. The reason for this is that previous studies on the SG knockout strain have been conducted on mice of C57BL/6 background, a strain with a defective mMCP-7 gene Copyright 2006 Biochemical Society C57BL/6 animals were also analyzed for mMCP-7 antigen. (C) Ear pinna were prepared from SG +/+ and SG -/-mice of DBA-1 background and were extracted with a high salt buffer, followed by immunoblot analysis for mMCP-7 antigen.