extfieldsofstudy: Engineering Medicine

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Angling for Uniqueness in Enzymatic Preparation of Glycosides

In the early days of biocatalysis, limitations of an enzyme modeled the enzymatic applications; nowadays the enzyme can be engineered to be suitable for the process requirements. This is a general bird’s-eye view and as such cannot be specific for articulated situations found in different classes of enzymes or for selected enzymatic processes. As far as the enzymatic preparation of glycosides is concerned, recent scientific literature is awash with examples of uniqueness related to the features of the biocatalyst (yield, substrate specificity, regioselectivity, and resistance to a particular reaction condition). The invention of glycosynthases is just one of the aspects that has thrust forward the research in this field. Protein engineering, metagenomics and reaction engineering have led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of glycosides. In this review, new examples from the last decade are compiled with attention both to cases in which naturally present, as well as genetically inserted, characteristics of the catalysts make them attractive for biocatalysis.

Introduction
In a brilliant, recently published analysis of the research-guided development in the field of biocatalysis during the last century, different authors recognized three historical waves of innovations that totally changed the field of biocatalysis to the present industrially accomplished level [1]. In a nutshell, while in the past limitations of an enzyme modeled the enzymatic process, today the enzyme can be engineered to be suitable for the process requirements. However this general bird's-eye view OPEN ACCESS cannot be specific for articulated contexts in which each single class of enzyme or selected enzymatic process is at the present state. In another similar general bird's-eye analysis, Riva identified a long wave of successes still far from reaching the end in biocatalysis [2], due to the difficulties encountered in the shift from 'classical' processes to biobased ones. It is clear that exploiting natural catalysts to obtain selective transformations of non-natural substrates is far from being fully explored; among many others, the cases represented by the new concept of 'third generation biorefineries' [2] (producing chemicals from biomasses), or by the new glycoside hydrolases and other enzymes found in marine environments [3] have re-fostered new research trends in the field. As a matter of example, although investigation into hemicellulases as biorefining enzymes has been slow, as reported in a recent analysis [4], xylan-related biocatalysis has continued to make steady progress in many areas, including the discovery and characterization of a wide range of hemicellulases. Talking more specifically about biobased glycosynthesis, these studies are opening new prospects for the use of pentose sugars as building blocks for engineered pentosides as non-ionic surfactants or prebiotic food/feed ingredients.
Carbohydrates are involved in a broad range of functions in cell living systems. Structural roles and energy storage as functions were recognized during the first half of the last century while attainments in glycobiology and glycochemistry, during the last twenty years, have further revealed that carbohydrate parts of biomolecules (glycoproteins, glycolipids, etc.) are involved in important biological functions mainly related to cell recognition events [5][6][7]. It should not be neglected that carbohydrates are important molecules also in the technological domain. Synthetic carbohydratecontaining polymers have a wide range of applications in medical biotechnology [8]. A number of novel dietary carbohydrates produced by enzymatic syntheses have been introduced into food technology during the last decade [9]. In innovative fine chemical manufacturing solutions, straightforward synthesis of products is of interest (e.g., chromophoric oligosaccharides of strictly defined structure as valuable tools for the kinetic analysis of hydrolytic activities and for characterization of new exo-or endo-glycosidases). Finally, in cosmetics, prodrug action of enzymatically glycosylated natural lipophilic antioxidants is currently under consideration.
In general glycosylation is considered to be an important and quite special method for the structural modification of a compound. It allows the conversion of a lipophilic compound into a hydrophilic one changing pharmacokinetic properties or creating drug delivery systems. It could also be generalized that in a glycoside, the type of aglycon determines the application: long alkyl chains allow glycosides to possess useful properties as surfactants and emulsifiers; aglycon based on unsaturated alkyl chains are said to be valuable, as glycosides, for fungal infections or as antimicrobial agents; glycosides of peptides and steroids are used in antitumor formulations and cardiac-related drugs, respectively; and glycosides of flavors and fragrances are used as "controlled release" compounds [10,11].
Sugar units have more than one site through which the chains are extended. Each of these sites frequently shows very similar reactivity, thus the masking of reactive centers by protecting groups is essential in order to direct coupling through the right position. For this reason protection and deprotection steps of functional groups are in use extensively in the arsenal of the synthetic carbohydrate chemist; moreover ensuring the correct stereochemistry of the glycosidic linkage formed entails additional difficulty. Carbohydrate related synthetic chemistry can still be considered one of the well-explored branches of organic chemistry and very rich in significant and spectacular successes, although important alternative biomethodologies for assembling glycosidic linkages are presently known and acknowledged. It is worth noting that in comparison with chemical methods, enzymatic glycosylation is particularly useful for the modification of complex biologically active substances, when generally harsh conditions or use of toxic (heavy metals) catalysts are undesirable. Enzymes may represent an imperative choice in fields such as agriculture and food or cosmetics where chemical strategies are not acceptable [12]. In a very recent report detailing different examples of enzymatic glycosylation of small molecules, the authors concluded that challenging substrates require tailored catalysts, and the progress in the field of enzyme engineering and screening of new catalytic activities are both expected to result in new applications of biocatalytic glycosylations in various industrial sectors [13].
The enzymes responsible for the synthesis of glycosidic linkage have been recognized as transglycosylases and named glycosyltransferases, specifying the glycosyl donor and the reaction product. These enzymes transfer sugar moieties from activated donors to specific acceptors, forming stereochemically specific glycosidic bonds, and are responsible in vivo for the synthesis of most cellsurface glycoconjugates, using eight common sugar nucleotides as activated donors (Leloir pathway). Sugar phosphates act as donors for other glycosyltransferases (non-Leloir pathway). Another widespread group of enzymes, named glycoside hydrolases (glycosidases), exists; they are involved in the carbohydrate metabolism being responsible for the hydrolysis of glycosidic linkages; they can act as exo-or endo-glycosidases and are involved in a series of important biological events such as energy uptake, in processes inherent cell wall metabolism, in glycan processing during in vivo glycoprotein synthesis, etc. Based on historical grounds, glycoside hydrolases were implicated in most experimental observations during the early studies into the biological synthesis of glycosidic linkages at the beginning of the last century. Hence, the concepts of enzymatically promoted synthesis by both hydrolysis-reversal and glycosyl transfer soon appeared [14]. By the end of the 1980s, several research projects [15] testified the importance of different and interesting glycoside hydrolases, especially from the marine environment; their main application was centered on the structural identification efforts that faced the complexity of oligosaccharide structures before the instrumental exploit of 2D NMR and MS spectroscopy.
Different wild-type glycosidases and their modified versions are enzymes deserving new expectations in research and development today. Significant progress has been made in recent years for the application of these enzymes: even while the major breakthrough was the invention of glycosynthases, protein engineering, metagenomics and reaction engineering led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of important glycosides. This review will compile different examples where glycoside hydrolases are the key enzymes in the process.

Natural Enzymes for the Synthesis of Glycosidic Linkages
In chemical terms, considering both hydrolytic or synthetic aspects of esterases, glycosidases, phosphatases, transglycosidases and peptidases, the enzymatic mechanisms are based on displacement reactions and could be grouped together. This line of thought proved to be highly productive in historical terms, allowing the collection and rationalization of the amount of mechanistic data especially for glycosidases and transglycosylases. The stereochemistry of the mechanisms of glycoside hydrolases was analyzed by Koshland [16] more than 60 years ago and allowed the classification of inverting and retaining enzymes according to the anomeric configuration found in the product with respect to that in the starting substrate. Very recently, it has become clear that other mechanisms have evolved, such as the one based on elimination [17]. In 2010, in an interesting review on diversity of catalytic base nucleophile of glycoside hydrolases, it was reported that a variety of systems are used to replace this function, including substrate-assisted catalysis, a network of several residues, and the use of non-carboxylate residues or exogenous nucleophiles [18].
Glycosyltransferases-mediated reactions are thought to proceed via an oxocarbenium-ion-like transition state as proposed for glycosidase reactions on the basis of solid structural, mechanistic and ab initio molecular orbital calculations data [19]. Glycosyltransferases are catalysts for natural glycosylation reactions, known as "Leloir" glycosyl transferases (GT). Glycoside phosphorylases (GP), requiring glycosyl phosphates and transglycosidases (TG), employing non-activated carbohydrates (e.g., sucrose), are additional examples of synthetic enzymes. However glycoside hydrolases (GH) can also be used for synthetic purposes under either kinetic (transglycosylation) or thermodynamic (reverse hydrolysis) control.
In this paragraph new examples related to the transfer of glycosyl residues between two oxygen nucleophiles are compiled with attention to those cases in which the natural characteristics of the catalyst make it attractive for biocatalysis (importance of molecular skeleton of substrates, yield, regioselectivity, resistance to particular reaction condition, etc.).

Interesting Transfer of Glycosyl Residues in Natural Enzymes for the Synthesis of Glycosidic Linkages
Hyaluronic acid, synthesized by hyaluronan synthases, is a biopolymer abundant in extracellular matrices that is degraded by hyaluronidases. Biocompatibility and biodegradability of this polymer and of related small derivatives are of great interest in pharmaceutics in a number of molecular devices for drug delivery. Bovine testicular hyaluronidase (BTH) is a commercially available hyaluronidase preparation that has long been considered a prototype of mammalian hyaluronidases. Presumably all mammalian hyaluronidases can catalyze hydrolysis as well as transglycosylation reactions of hyaluronic acid fragments ( Figure 1). In the case of BTH, the hydrolysis is favored at acidic pH values, while transglycosylation occurs preferentially at neutral pH and at low NaCl concentrations. The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 has facilitated the first detailed analysis of the enzymatic reaction products for these two enzymes. HA hexasaccharide, which is generally accepted as being the minimum substrate of BTH, is not a substrate of recombinant human PH-20 and Hyal-1 as recently demonstrated [20] although BTH and PH-20 belong to the same type of hyaluronidase. Interestingly, HA octasaccharide can be used as the substrate of Hyal-1 at pH 3.5. The substrate was converted quickly at concentration between 25 µM to 1 mM; above 1 mM weak substrate inhibition was observed. The study of transfer reactions, selectivities and yields are all features of interest for a possible use of these enzymes in biocatalytic steps for the manipulation of important biomolecules [21]. Additional new examples of this enzyme can be derived from other environments: the venoms of two classes of fish, freshwater stingray (members of the genus Potamotrygon) and stonefish (members of the genus Synanceia), contain, along with proteinaceous toxins, also hyaluronidases. These proteins are considered as spreading factors that facilitate the tissue diffusion of toxins by degrading hyaluronan; owing to this quick action it can possess very interesting features for biocatalysis [22].
Sensitive and reliable enzymatic tools for the analysis of glycan chains are needed. The O-linked glycans are attached to serine or threonine through the GalNAc residue at the reducing end. The hydrolysis of the O-glycosidic α-linkage between GalNAc of the disaccharide Gal-β-1,3-GalNAc-and threonine or serine, is catalyzed by endo-α-N-acetylgalactosaminidase (EC 3.2.1.97). Most of the enzymes of this type are strictly specific for the disaccharidic structure and have no action on substrates with longer or different glycosyl chains. Using the protein sequence of a known endo-α-GalNAcase from B. longum, four potential sequences were found from BLAST (Basic Local Alignment Search Tool) search. Cloned and expressed proteins were purified and characterized [23]. Substrate specificity was investigated on aryl substrates as indicated in Figure 2 or by using natural glycoproteins. All three new enzymes are active on Core 1 substrate (Gal-β-1,3-GalNAc) while only two of them (EngEF and EngPA) were active after 24 h on Core 3 disaccharide (Glc-β-1,3-GalNAc). Interestingly, these enzymes acted also as transglycosylating agents; when reacted with simple alkanols (from methanol to nonanol) as acceptors, Core 1 and Core 3 acted as donor disaccharides in test reactions. Although the yields as judged by TLC analysis were low (at 0.8-1.6 mM donor and 13% v/v alkanols), positive reactions were observed up to 4-5 alkanol carbon atoms. As stated by the authors in the concluding remarks, the action of EngEF and EngPA enzymes acting on Core 3 in addition to Core 1 O-glycans could make these enzymes powerful tools for the release of O-glycan sugars from glycoproteins. More importantly they can also be used as templates in future protein engineering experiments for a possible creation of endo-α-GalNAcases capable of acting on O-linked glycans, regardless of their sugar composition. One of the last outstanding results in transglycosylation reactions in the last decade is, without any doubt, the recognition that endo--N-acetylglucosaminidase can transglycosylate a large oligosaccharide onto various glycosyl acceptors. In 2001, the Shoda group reported on the synthesis of a novel disaccharide possessing a 1,2-oxazoline moiety and tested it with a series of enzymes for transglycosylation activity. Typical oxazoline substrate 1, as depicted in Figure 3, reacted with Endo-M or Endo-A from Mucor hiemalis or A. protophormiae, respectively and the acceptor GlcNAc--1-O-pNP for the synthesis of a trisaccharidic derivative in high yield [24]. These interesting biocatalysts (Endo-M or Endo-A), which belong to GH family 85 were inactive when they have to use acceptors capped with an α-1,6-fucose (1,6-fucosyl-GlcNAc derivative as acceptors for transglycosylation). But more recently, the Huang group, screening various endo-β-N-acetylglucosaminidases using appropriate synthetic oxazoline donors and compound 2, Figure 3, as acceptor substrate, found that endo-β-Nacetylglucosaminidases from Flavobacterium meningosepticum (including Endo-F2 and Endo-F3), were able to glycosylate α-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex type N-glycopeptides [25]. The product(s) were isolated in high yield by HPLC and this efficiency of transglycosylation is quite impressive, given the fact that only two-fold of the donor substrate was used.
Starting from the known consideration that extracellular glycosidases from fungi, (used to access low molecular weight sugars by their hydrolytic action on polymeric substrates), must possess high flexibility in substrate specificity, the group of Kren recently investigated new enzymes in which the 4-hydroxy moiety of the pyranose ring of the substrate is not essential for binding to the enzyme active site. In their report they emphasize also the transglycosylation activity of β-N-acetylhexosaminidase from Talaromyces flavus. The results reported on the production of three novel 4′-deoxy-disaccharides prepared in high yields (52% total disaccharide fraction) starting with phenyl 2-acetamido-2,4-dideoxy-substrate. The conditions of the transglycosylation reaction were first optimized on an analytical scale by varying the concentrations and ratios of the reaction components and identifiying the following reaction conditions as the most efficient: 75 mM donor, 300 mM acceptor, incubation for 5-6 h at 35 °C [26]. The enzymatic formation of -D-fucosides is hardly described in the scientific literature. An extraordinarily broad substrate specificity for both hydrolysis and transglycosylation was exhibited by a glycosidase isolated from the China white jade snail. Acceptor specificity for monosaccharides and transfer efficiency have both been investigated for this promising enzyme [27] and from the results obtained, the authors indicated a very high transfucosylation efficiency using p-nitrophenyl derivative of -D-fucose at 10 mM and acceptors (at 20-100 mM) such as glucose (88% yield) and xylose (93% yield); the interglycosidic linkage formed with glucose is -1,6 thus they proposed this biocatalyst as a useful candidate for disaccharide synthesis. The Fuc--Xyl disaccharide formed in other reactions is a building block for the synthesis of asterosaponins of marine origin, although in natural compound the interglycosidic linkage is different (Fuc--(1-2)-Xyl) [28].
In screening a suitable biocatalyst for galactosylation of nucleosides, Ye et al. very recently found that -galactosidases from Kluyveromyces lactis (Sigma, USA) and a crude glycosidase extract of apple seeds, had high hydrolytic activities toward oNPGal, but could not catalyze transglycosylation reactions between oNPGal and 2-deoxynucleosides [29]. Indeed, only very few reports have appeared in the literature in the last 10 years leading with successful glycosidase-mediated nucleoside glycosylation: (i) after low-yielding results using -galactosidase from Aspergillus oryzae [30], the first successful approach, which appeared in 2007, was (ii) the convenient synthesis of -galactosyl derivatives of antiviral and anticancer nucleosides, utilizing the purified -galactosidase activity from the hepatopancreas of Aplysia fasciata [31] and the second valid method is (iii) the regioselective galactosylation of floxuridine (FUdR) catalyzed by a commercial -galactosidase from bovine liver with a high yield (75%) and an excellent 5--regioselectivity (>99%) [32] using o-nitrophenyl -D-galactoside as glycosyl donor. Such desirable products were synthesized with satisfactory yields (41-68%) and moderate to high 5-regioselectivities (87-100%), also by using the crude enzyme extract.
In the hepatopancreas and visceral mass of the mollusc Aplysia fasciata a wide range of glycoside hydrolases (α-glucosidase, -galactosidase, -glucosidase, -mannosidase and others) were found and successfully used for the hydrolysis and synthesis of glycosidic bonds. The formation of α-Doligoglucosides from maltose (up to tetra-and pentasaccharides) was studied in detail [33] and it was also demonstrated that the enzyme was able to form mono-and di-and poly-glucosides of different externally added acceptors [3 and references cited therein]. This activity has been applied in the very recent chemoenzymatic synthesis of α-6-sulfoquinovosyl-1,2-O-diacylglycerols, a class of natural lipids that have attracted biomedical attention as possible antitumors, antivirals, and immunomodulators. This synthesis started with the enzymatically controlled transfer of glucose from maltose (10:1 molar ratio) to (rac)-1,2-O-isopropylidene glycerol. The 1,2 glycerol acetonide conversion of 59% in 29 h was obtained and reaction products were exclusively 3-α-glycosyl derivatives of 1,2-O-isopropylidene glycerol. In particular 3-α-glucosyl-1,2-O-isopropylidene glycerol was produced as the major component (30% yield) together with a mixture of di-and tri-saccharide analogues (23 and 6% yields, respectively). Under similar conditions the transglycosidase of Aspergillus niger and the α-glucosidase of Bacillus stearothermophilus gave 3-α-glucosyl-1,2-O-isopropylidene glycerol with yields below 2%. This comparison suggests that marine enzymes may offer a viable option for the synthesis of different types of glycolipids [34].
There are other glycoside hydrolase activities that are hardly used in synthesis. A thermostable α-L-arabinofuranosidase was reported in 2002 for its ability to perform transarabinosylation reactions for the synthesis of different α-L-arabinofuranosides of various alcohols. The same enzyme was later reported as a useful biocatalyst for the synthesis of p-nitrophenyl α-L-arabinofuranosyl-(1-2)-α-Larabinofuranoside, p-nitrophenyl -D-xylopyranosyl-(1-2)--D-xylopyranoside, p-nitrophenyl -Dxylopyranosyl-(1-3)--D-xylopyranoside and benzyl α-L-arabinofuranosyl-(1-2)-α-D-xylopyranoside. Such a disaccharidic motif could be used as the reference compound for the analysis of hemicellulase action and for raising antibodies to well-defined motifs for immunochemical-based analyses of plant cell walls [35]. The same group reported also the synthesis of galactofuranosides using the above catalyst [36]. -D-galactofuranose is a cell wall constituent in several pathogenic species including Mycobacterium tuberculosis and leprae, agents of tuberculosis and leprosy, respectively. The synthesis of galactofuranose analogues acting as inhibitors of the biosynthetic enzymes (UDP-galactopyranose mutase and galactofuranosyl transferases) or the synthesis of galactofuranosides that could be used for the elaboration of vaccines, is important for this aspect. Authors reported also on the chemical synthesis of p-nitrophenyl galactofuranoside used as donor and the low yield obtained (16%) could act as an additional center of interest for the synthetic aspect of this kind of enzyme.
An interesting case of the effect of solvents in transglycosylation reactions has been studied recently for the synthesis of N-acetyl-D-lactosamine, Gal--1,4-GlcNAc (LacNAc), catalyzed by the -galactosidase from Thermus thermophilus (TTP0042). The authors studied the percentages of all products formed (self-condensation products, hydrolytic originating galactose and transfer regioisomers). The low yield (about 20%) of LAcNAc when the reaction is performed in buffer is increased by up to 91% when the process takes place in the presence of glycerol based solvents. According to conformational studies of the enzyme structure by circular dichroism and fluorescence, the authors concluded that in the presence of these solvents the enzyme modifies secondary and tertiary structure and this may also be the cause of some additional regioselectivity changes observed [37]. Unfortunately no molecular details were furnished, indeed no clear relationship between the very different solvent structures used and the regioselectivity change was envisaged.
A new and interesting fungal diglycosidase was isolated from Acremonium sp. It has an α-rhamnosyl--glucosidase activity with transglycosylation potential of the entire disaccharide (rutinose) moiety from natural products to different acceptors. This enzyme allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor with a 16% yield regarding the sugar acceptor [38]. Despite the low yield, the fluorogenic substrate formed, 4-methylumbelliferyl rutinoside (Figure 4), which is not commercially available, is important for the study of diglycosidases since it allows the detection of enzymes specific for rutinose mobilization. Although the activity of this enzyme could seem quite specific for a broad interest, we must bear in mind its potential use for industrial processing of plant-based foods and the ability to transglycosylate rutinosyl units from abundant and inexpensive by-products of citrus industry.

Engineered Enzymes
From 2004 to 2013 PubMed indexed 78 hits having the words glycosynthase(s) in the title, including review articles that accounted for ca. 20%. Similar figures can be found in Scopus and in Web of Knowledge. Using data published up to the mid-term of 2003, Perugino et al. compiled a review [39] accounting for 11 different glycoside hydrolases from bacteria, eukarya and archaea, that were modified as efficient glycosynthases. In 2012, in an update report from the same lab [40] a quadrupled value was found. Although these lists were declared not exhaustive, a similar number is also reported in the review of 2011 from Wong labs [41]. Indeed a number of excellent reviews periodically survey the most recent achievements in the field of glycosynthases from different perspectives, such as their production and application to the synthesis of glycoconjugates. The obvious success is due to quantitative yields that can be reached in reactions using these active-site modified glycoside hydrolases. The glycosynthase approach is certainly of great help while keeping molecular diversity offered by different natural glycoside hydrolases, including features such as resistance to temperature, organic solvent, etc., making this aspect very interesting for the exploitation of these enzymes in biocatalysis. A survey of all these examples will not be repeated here, where instead just one case of a spectacular glycosphingolipids synthesis is reported below.
The focus in the remaining part of this paragraph, dedicated to engineered enzymes, is on those cases where genetic modifications could help in different manner than increasing yield by glycosynthase philosophy.

Preparative Glycosphingolipids Synthesis Operated by Glycosynthase
Pharmacological interest for glycosphingolipids, a class of glycolipids based on the aminodiol sphingosine, is high since these compounds have numerous biological functions in processes of human physiology [42]. Gangliosides are a subclass of glycosphingolipids containing also sialic acid in the oligosaccharide moiety, composed of different sugars. Certain gangliosides are involved in viral infections mechanisms. Commercial sources for glycosphingolipids rely on isolation of compounds from natural sources, such as bovine brain and canine blood. This is impractical for large-scale preparation, as well as posing risks for contamination and requiring great effort for extensive purification. A spectacular example of application of glycosynthase technology for high yield production of different gangliosides has been recently reported [43]. The work is based on the transformation of a natural hydrolytic enzyme, the endoglycoceramidase II (EGC II) from Rhodococcus strain M-777, in a glycosynthase. This glycosynthase version of the enzyme with Ala, Ser or Gly as substituents of active site nucleophile, is not capable of performing hydrolysis, but it can react in synthetic mode using α-anomer of different oligosaccharidic fluorides (di-to pentasaccharides) in the presence of acceptors such as D-erythro-sphingosine or sphingosine analogues ( Figure 5). Yields from 70% to 100% were obtained for the lyso product on 300 mg scale. Reactants so structurally different could pose problems, but the solubility of the hydrochloride salt of the sphingosine in aqueous buffer (25 mM after sonication at 37 °C ) is considered by the authors a benefit for possible molecular diversity that can be obtained with successive acylation steps with structurally diverse acyl chains. In fact, N-acyl substituents in ceramides affects biological activity of these compounds to some extent, and the acyl substituent has also been identified as a convenient position for conjugation of non-lipid substituents, including fluorescent tags [44].

Engineering Glycoside Hydrolases not at the Active Site
Engineering the active-site environs is a technique that has been used to enhance the transglycosylation activity of glycosidases and to modify other features of interest in biocatalysis. When structural architecture of the active site is not known, a site-specific "randomization" approach could be used in which an amino acid residue near the catalytic site can be replaced by each of all other amino acids to screen mutant enzyme(s) of interest. This strategy was used to obtain α-glucosidase variant(s) for increasing the production of theanderose, a trisaccharide obtained from sucrose by α-glucosylation to the C6 of glucose. An amino acid residue (Gly273 or Thr272) near the putative catalytic site (Glu271) of this Bacillus α-glucosidase was replaced by all other naturally-occurring amino acids thus increasing the transglucosylation activity. The highest specificity for theanderose formation (i.e., the highest content of theanderose in the reaction product) was obtained with the isoleucine containing mutant (T272I), which showed 1.74 times higher productivity (per sucrose-hydrolyzing unit) of theanderose than that of the wild-type enzyme. The authors concluded, however, that elucidation of three-dimensional structures should help to understand the details of the mechanism eliciting the specificity obtained [45]. More recently, the transglycosylation/hydrolysis ratio was shown to be 3-and 8-fold increased with two mutants of a different enzyme, the Thermotoga neapolitana -glucosidase. The asparagine mutant (N291T) of this enzyme showed also altered regioselectivity. In particular TLC analysis of the transglycosylation products indicated that while the mutant retained its β-1,3 regioselectivity, β-1,4 and β-1,6 selectivities were lost when pNPG and arbutin were used as a donor and acceptor, respectively [46]. In another case, Feng et al. in 2005 described directed evolution applied to the -glycosidase of Thermus thermophilus to increase the ability of this enzyme in transglycosylation reaction. The most efficient mutations of phenylalanine and asparagine (F401S and N282T), were located just in front of the acceptor subsite and the authors suggested that repositioning of the glycone in its subsite together with a better fit of the acceptor in the acceptor subsite, might favor the attack of a glycosyl acceptor in the mutant at the expense of water; this conclusion was based on molecular modeling techniques. They also concluded that their results suggest that directed evolution of the glycosidases in transglycosidases could be an alternative to the glycosynthase strategy; in fact, for certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative [47]. However when the asparagine (N282T) mutant was analyzed with external added acceptor pNPGlcNAc using oNPGal as donor, the NMR study of kinetic formation of transglycosylation products showed that those due to self-condensation (Gal1-3Gal-oNP and Gal1-6Gal-oNP) are kinetically favored over the condensation product Gal1-4GlcNAc-pNP, suggesting that oNPGal is a better acceptor than pNPGlcNAc; competition between self-condensation and condensation could be responsible for the moderate yield of the desired product Gal1-4GlcNAc-pNP. Based on the analysis of catalytic context, the authors studied in silico the complex alanine mutant-acceptor (A221W)/pNPGlcNAc establishing that the docking of the acceptor is not perturbed by the mutation, compared to the WT case. Donor docking was also studied with A221W mutant complexed with oNPGal to analyze precisely the perturbation as compared with WT. In WT enzyme there is enough room for oNPGal in the acceptor sites allowing the self-condensation reaction. In the A221W mutant, a drastic steric conflict occurs between the galactose ring and the tryptophan substituting alanine and this effect was seen by the authors as the most promising for preventing the self-condensation reaction. The mutation A221W was thus introduced by directed mutagenesis. The analysis of products and relative yields of this double mutant N282T/A221W shows that these results are consistent with the MM calculations. The authors proposed their approach as convenient in that relatively stable activated sugars can be used [48]. While this work shows the value of a rational approach to eliminate the side effects of transglycosylation reactions, a thermophilic glycosynthase from Sulfolobus solfataricus was indeed shown to act in presence of external formate on stable activated sugars such as oNP-glyco donors [49].
A random mutagenic approach coupled to a screening procedure was applied in another thermophilic case in nature. The screening was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose, -D-galactopyranosyl-(1-4)-D-sucrose) formed in presence of sucrose, thus providing mutant enzymes possessing improved synthetic properties for the transglycosylation [50]. The application of thermostable -galactosidases such as the -galactosidase BgaB from Geobacillus stearothermophilus is of interest for transgalactosylation because at higher temperatures higher lactose concentration can be used to favor the synthesis.
A complete change of product profile of the reaction was observed for one of the mutants obtained by site directed mutagenesis of α-amylase of Bacillus amyloliquefaciens. The Val289 residue substituted with tyrosine showed less than 15% activity compared to the wild-type, but it acquired transglycosylation activity, producing longer oligosaccharides. The analysis of all mutants produced led to the conclusion that changes in the hydrolytic property of α-amylase may be due to factors like the geometry and electrostatics of the environment around the active site [51].
It could be of interest in this paragraph to mention the development of methodologies for the screening of large libraries of mutant enzymes. In a paper of 2009 Konè et al. reported on digital screening methodology as a system dedicated to the screening for sugar-transfer activity [52], which gave great impetus to the study of glycosidases with efficient transglycosidases activity as an alternative to glycosyltransferases or glycosynthases. Recently, from genomic and metagenomic program results, protein sequences with unknown functions are increasing, thus effective screening methodologies have become an important aspect of this research. In the field of polysaccharide degrading enzymes, a profiling method reported in 2012 by the Helbert group is worth noting. Polysaccharidases are important enzymes that are used as specific tools to improve conversion of lignocellulosic biomass into sugar monomers prior to ethanol production, for: degradation of microbial polysaccharides, obtaining bioactive materials, for structural determination of unknown complex polysaccharides structures, etc. The profiling strategy is based on a series of filtrations necessary to eliminate any reducing sugars not directly generated by enzyme degradation. After enzymatic action, filtrates are assayed with a ferricyanide solution to reveal the reducing sugars produced by glycoside hydrolases or polysaccharide lyases; however matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), presenting several unique advantages for the structural characterization of degradation products of carbohydrates, is also considered as providing an effective methodology [53,54].

Conclusions
Although not exhaustively catalogued in this review, the scientific literature of the last decade, with regard to the process of enzymatic preparation of glycosides, enables the reader to conclude that research for uniqueness, in terms of enzymatic features, is still active. This is in contrast with the tempting generalization about enzymes as plastic biomolecules fully receptive to be engineered by appropriate changes, although this plasticity has been successfully demonstrated in some examples [1]. The list of naturally "unique" enzymes cited in this review includes examples found among the most promising subjects of biocatalytic applications: (i) hyaluronidases for producing and/or transferring hyaluronic oligosaccharides of pharmaceutical interest or (ii) disaccharidases such as endo-α-Nacetylgalactosaminidase (for the analysis of glycan chains) and (iii) the fungal rutinosidase used in the synthesis of related fluorogenic derivative of 4-methylumbelliferone, are of specific interest. Among engineered enzymes, even though glycosynthases have to be seen as the most successful approach to reach quantitative transfer yields in transglycosylations, engineered enzymes examples with mutations only in the active-site environ (and not at active site aminoacids), demonstrated that biocatalytic features are well manipulated also in this way, thus enlarging competence to regioselectivity and or specificity for heteroacceptors and not only to reaction yield as devised for glycosynthases.
This overview should increase the interest from biocatalyst practitioners towards both naturally unique enzymes and for infrequent mutations capable of inducing biocatalytically interesting features.


extfieldsofstudy: Biology Medicine

text:
Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-β) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-β. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-β induced type II EMT in vivo.

Introduction
Epithelial-mesenchymal transition (EMT) is a biological process that allows a polarized epithelial cell to undergo several biochemical/morphological changes to gain a mesenchymal phenotype [1]. The process was first described by Elisabeth Hay who also depicted the basic differences of these cellular actions during embryogenesis and tumorigenesis [2]. Since then three subtypes of EMTs have been distinguished with different functional consequences. Besides epithelial-mesenchymal transition during embryogenesis (type I) and tumorigenesis (type III), type II EMT is associated with wound healing, tissue regeneration and organ fibrosis [3], [4]. It has been demonstrated that upon inflammation many cells (monocytes/macrophages, fibroblasts) can trigger type II EMT through secretion of growth factors such as transforming growth factor-beta (TGF-b) or epidermal growth factor (EGF). Most prominent among these cells are the macrophages and activated resident fibroblasts that accumulate at the site of injury and release these growth factors [1], [2], [5]. TGF-b was first described to induce EMT via Smad 2/3dependent pathway and meanwhile it became evident that the cellular actions of the cytokine can be modulated and defined by other Smad-independent signaling pathways like the MAP kinase pathways [6]- [8].
Recently, estrogen receptor alpha (ER-a) has been suggested as another player in the molecular mechanism of EMT [9]- [11]. For long, estrogen receptors (ER-a, ER-b) have been considered exclusively as transcription factors acting inside the nucleus [12], [13]. However, the discovery of its membrane-associated form and the ER-mediated transcription in the absence of its ligand generally changed this concept [14], [15]. The theory of a hormon-independent ER-a activation that can serve as a mechanism to amplify growth factor pathways [16] has also been accepted by now. It has been proved that a small percentage of ERs (5-10%) reside in the cell membrane and can elicit both genomic and non-genomic responses by activating multiple protein kinase cascades that include MAPK, protein kinase C, Src kinase and PI3K [17]- [23].
ER-a and TGF-b have opposing roles in cell proliferation and differentiation of epithelial cells. Their regulatory pathways intersect and ER-a blocks the TGF-b pathway at different cellular levels inside the nucleus as well as in the cytoplasm and plasma membrane. Both transcription factors have a prominent role in maintaining a controlled signaling that is essential for cell and tissue homeostasis and both act in a cell-specific and contextdependent manner [24]- [25].
In our previously published papers we showed that mesothelial cells that form a continuous squamous epithelial layer on the two sides of the mesentery undergo epithelial-mesenchymal transition (EMT) upon Freund's adjuvant treatment [26]. It has also been proved that upon inflammatory stimuli mesothelial cells can assume a macrophage character demonstrated by the expression of the macrophage marker, ED1 [27] and they can serve as a source of activated macrophages during inflammatory events [28]. Besides this finding, it is also known that macrophages express ERa [29], [30]. These data together led us to examine whether mesothelial cells express ER-a and if so, what is the subcellular distribution of the receptor. We examined the morphological and biochemical changes of mesothelial cells under inflammatory stimuli in vivo. This study reports for the first time that ER-a is expressed by the rat mesenteric mesothelial cells and an inverse correlation between the level of ER-a expression and the peritoneal secretion of TGF-b can be observed in response to Freund's adjuvant treatment. The present results show that ER-a is associated with caveolin-1 protein and the receptor is presumably internalized via caveola-mediated endocytosis. Our morphological observations and biochemical data together indicate that ER-a and its caveola-mediated endocytosis may play role in TGF-b induced type II epithelial-mesenchymal transition.

Ethics Statement
All rat experiments were carried out in accordance with the recommendations in the Guide for the use of Adjuvants in Research of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Amherst [31] and Semmelweis University's Institutional Animal Care and Use Comittee. All efforts were made to minimize suffering.

Material and Treatment
Rat mesentery (peritoneum of small intestine) was isolated from control and Freund's adjuvant (Sigma, Steinheim, Germany) treated Sprague-Dawley male and female rats (200-400 g). 1 ml complete Freund's adjuvant was injected into the peritoneal cavity. One day (D1), 2 days (D2), 3 days (D3) and 5 days (D5) following intraperitoneal injections the animals were sacrificed by decapitation and mesentery samples were removed.

Collection of peritoneal fluid, isolation of mesothelial cells
To determine the secretion level of TGF-b, peritoneal fluid (PF) samples were collected from both male and female animals by washing the peritoneal cavity with 2 ml phosphate-buffered saline (PBS). The PF samples were centrifuged with 1000 rpm, for 10 min at 4uC and the supernatants were stored at 220uC until use for immunoblotting.
For isolating mesothelial cells, mesentery was removed and placed into 0,2% collagenase diluted DMEM tissue culture medium (Sigma) for 45-50 minutes at 37uC. The solid remnants (adipose tissue, connective tissue) were removed and samples were washed three times in PBS and centrifuged with 1000 rpm, for 10 min at room temperature. The pellets were then placed in liquid nitrogen for 10 minutes and stored at 280uC until RNA isolation for quantitative RT-PCR.

RNA isolation and quantitative RT-PCR
Total RNA was extracted with RNeasy tissue mini kit (QIAGEN Inc., Chatsworth, CA). For qualitative and quantitative analysis of RNA preparations, RNA 6000 Nano Chip kits in an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) was used according to the manufacturer's instructions. Following RNA isolation, RNAs obtained were transcribed into cDNAs using SuperScript TM III First-Strand Synthesis SuperMix (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. From each samples, 0.5-1 mg of total RNA was converted into cDNA. Real-time PCR was performed using Taqman Universal Master Mix II, No UNG and the 7900HT Real-Time PCR System (Life Technologies, Carlsbad, CA), with the following parameters: 50uC for 2 min and 95uC for 10 min, followed by 40 two-step cycles at 95uC and at 60uC for 1 min. Applied Biosystems pre-designed TaqManH Gene Expression Assays were used for real-time PCR (Il6 Rn00561420_m1, Il1a Rn00566700_m1, Il1b Rn00580432_m1, Esr1 Rn00664737_m1, GAPD.4352338E, Esr2 Rn00562610_m1, Gpr30 Rn00592091_s1 and B2m Rn00560865_m1). The qRT-PCRs were executed in triplicate using TaqMan probe synthesized by Applied Biosystems). SDS 2.3 (Applied Biosystems, Foster City, CA) and RQ manager 1.2 softwares (Applied Biosystems, Foster City, CA) were applied for calculation of the threshold cycle (Ct) values in each sample.
Expression level was calculated by the ddCt method, and fold changes (FC) were obtained using the formula 2 -ddCt . Computed internal control corresponding to the geometric mean of Ct values of the two houskeeping genes (GAPD and B2m) was used for the ddCt calculation. Gene expression levels at different time points were compared to controls using ANOVA and t-test with a statistical software SPSS version 13 (SPSS Inc., Chicago, IL, USA).

Antibodies
Primary antibodies were polyclonal anti-caveolin-1 antibody

Western blotting
The protein contents of the supernatants from peritoneal fluid samples were determined by the Bradford method [32] and diluted to a concentration of 1 mg/ml. Afterwards the samples were solubilized with reducing Tris-SDS buffer (Tris 0.5 M pH 6.8, 10% glycerol, 2% SDS, 0.00125% bromophenol blue, 0.5% mercapto-ethanol) and heated at 95uC for 4 min. 15 ml per well from the samples were loaded onto an 10% Acrylamide/Bis gel and separated by electrophoresis. After separation, proteins were transferred onto a nitrocellulose membrane (Amersham, GE Healthcare Biosciences, Pittsburgh) in a buffer containing Tris-Glycine pH 8.3, 0.1% SDS and 20% methanol.
Aspecific reactions were blocked at room temperature for 2 h in PBS-Tween (0.5 M PBS, Tween 0.05%) containing 5% skim milk powder. The membranes were then incubated with polyclonal anti-rabbit TGF-b primary antibody (Cell Signaling Technology Inc., Beverly, MA) diluted 1:1000 at 4uC for overnight. After washing in PBS-Tween the membranes were treated with speciesspecific peroxydase-conjugated secondary antibody (Amersham, GE Healthcare Biosciences, Pittsburgh) for 1 h at room temperature. The labeled protein bands were visualized by the ECL Plus chemiluminescence method and developed onto high performance chemiluminescence film (Amersham, GE Healthcare Biosciences, Pittsburgh). Relative optical densities were measured using the ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland) and the results of four independent experiments were compared and statistically analyzed. The significance was tested by the ANOVA method and Tukey's HSD test.

Tissue fixation for light and electron microscopy
Mesentery was isolated from both control and Freund's adjuvant-treated animals. The samples were fixed either in a 1:1 mixture of 2% glutaraldehyde (GA in 0.2 M cacodylate buffer) and 2% OsO 4 in distilled water (30 min, on ice) or freshly prepared 4% paraformaldehyde (PFA) in 0.1 M PB (2-4 h, room temperature). The samples were washed in cacodylate buffer and PBS, respectively and the adipose tissue was removed from the mesentery. The GA-Os fixed material was proceeded to electron microscopic embedding, while the PFA-fixed samples were used for immunocytochemistry.

Electron Microscopy
The GA-Os fixed samples were washed in 0.1 M cacodylate buffer three times, dehydrated with ethanol and stained with 1% uranyl acetate (in 70% ethanol for 1 h, room temperature) prior to araldite embedding. Ultrathin sections were contrasted with uranilacetate and lead citrate. The samples were analyzed in a Hitachi H-7600 (Tokyo, Japan) transmission electron microscope.

Preparation of semithin and ultrathin cryosections
The PFA-fixed samples were stored in 1% formaldehyde (in 0.1 M PB) at 4uC until further processing. For semithin cryosectioning and immunolabeling, the fixed samples were washed twice in PBS, once in 0.02 M glycin/PBS and infiltrated gradually with gelatine solutions of increasing concentrations (2%-5%-12% in PB) at 37uC for 30 min each. The samples were oriented in liquid gelatin and cut into small blocks. For cryoprotection, the blocks were infiltrated with 2.3 M sucrose at 4uC overnight and afterwards mounted on metal pins, frozen and stored in liquid nitrogen. For preparing semithin and ultrathin cryosections we used Leica Ultracut S ultramicrotome equipped with cryo-attachment (Vienna/Austria). The pickup solution was a 1:1 mixture of 2.3 M sucrose and 1.8% methylcellulose.
Immunolabeling for light and electron microscopy 0.5 mm semithin sections mounted on microscopic slides were incubated with 0.02 M glycine in PBS for 15 min and then they were blocked in PBS containing 1% BSA. Primary antibodies were applied in 1% BSA containing buffer in a humidified chamber at 4uC (overnight). Polyclonal anti-caveolin-1 and polyclonal anti estrogen-receptor alpha antibodies were used at the dilution 1:200 and 1:100, respectively. After washing with PBS, this was followed by a 1 h incubation with the secondary antibody in a humidified chamber at room temperature. Sections were then rinsed with PBS, stained with DAPI, placed under coverslips and visualized in a Bio-Rad (Ontario, Canada) Radiance 2100 Rainbow confocal microscope.
For cryosectioning and immuno-EM, the fixed tissues were further processed as described [33]. Ultrathin cryosections prepared at 2100uC were transferred to copper grids by pickup with a 1:1 mixture of 2.3 M sucrose in PBS and 1.8% methylcellulose. For immunogold labeling, the grids were incubated first on 2% gelatin/PBS at 37uC, blocked with 0.02 M glycine/PBS and subsequently incubated with primary antibodies followed by protein A-gold. The sections were contrast stained with 2% uranyl acetate/oxalate, pH 7, followed by 0.4% uranyl acetate pH 4 and 1.8% methyl cellulose and dried. The cryosections were analyzed in a Hitachi H-7600 (Tokyo, Japan) transmission electron microscope at 80 kV.

Ultrastructural and biochemical evidences of type II EMT in mesothelial cells in vivo
In untreated, control animals mesothelial cells form a continuous simple squamous epithelial layer on both sides of the mesentery. As observed in the electron microscope, the cytoplasm of these cells was rather poor in cell organelles and the cells were dominated by the nucleus. A continuous basal membrane separates the mesothelial cell layer from the underlying connective tissue and maintains the integrity of the mesothelial layer. Caveolae (caveolin-containing lipid rafts seen as flask-shaped invaginations of the cell membrane) were abundantly present on the basolateral and apical surfaces of the cells (Fig. 1A).
Our previous light microscopical data revealed that mesothelial cells lose their epithelial character (decreased cytokeratin, Ecadherin expression) upon Freund's adjuvant treatment and obtain a mesenchymal phenotype by expressing vimentin [26]. The inflammatory stimuli changed remarkably the ultrastructure of mesothelial cells as well. These changes culminated on the second to third day (D2/D3) after treatment. From the fifth day (D5) on the tissue started to recover and the repairment was morphologically accomplished by the eleventh day (D11). Characteristic changes were the disintegration of basal membrane and transformation of the squamous mesothelial cells into individual cuboidal-shaped cells in two days after treatment (Fig. 1B). By the fifth day, cells developed numerous lamellar processes and became spindle-shaped (Fig. 1C). The cytoplasmic compartments were more prominent: an increased number of mitochondria, polyribosomes, numerous vesicles and a growing number of multivesicular bodies (MVBs) could be observed in parallel with the inflammatory events of the surrounding tissue. A striking observation was the presence of thickened, electron-dense domains on the limiting membrane of MVBs with a fine coat of medium electron density on the cytosolic side (Fig. 1D). These specific membrane domains (referred from now on as MVB plaques) disappeared by the later stages of the MVB maturation process and could not be observed in the membrane of intermediate forms between MVB and lysosome (Fig. 1E).
In addition to ultrastructural changes, it was of primary importance to see if Freund's adjuvant treatment induces inflammatory responses in our in vivo system at a molecular level as well. To verify this, we determined the expression levels of proinflammatory cytokines in mesothelial cells as they are well-known to be involved in immune responses and inflammatory processes. The results of quantitative RT-PCR showed that mRNA expression levels of interleukin type 1alpha and type 1beta and also interleukin 6 increased in mesothelial cells by Freund's adjuvant treatment (Fig. 2A). The elevated mRNA levels of these cytokines correlated with the time-scale of the observed morphological changes during the inflammatory events: expression levels of mRNAs had a peak on D3 followed by a significant downregulation that could be observed from the fifth day indicating the termination of the inflammatory response. Our Western blot data showed that TGF-b was secreted into the peritoneal cavity (Fig. 2B, C) and the secretion of the cytokine was in accordance with the morphological changes and with the

Estrogen receptor alpha (ER-a) is expressed in mesothelial cells
Since mesothelial cells can serve as one of the sources of activated macrophages upon Freund's adjuvant treatment and it is known that macrophages express ER-a, the question arose whether mesenteric mesothelial cells also express the molecule? Our present results show that ER-a is present in mesothelial cells both under steady state and inflammatory conditions. As control cells were flat and the cytoplasm formed a thin rim around the nucleus, it was difficult to define the exact nuclear, cytoplasmic or plasma membrane (PM) localization of the marker with light microscope (Fig. 3A). The morphological changes of the cells upon inflammation enabled us to better detect the localization of ER-a. Confocal microscopical results after immunofluorescence detection of ER-a showed that labeling appeared in the nucleus, cytoplasm as well as in the plasma membrane and on the consecutive days after treatment the intensity of the labeling increased ( Fig. 3B-D). In those mesothelial cells that had entirely lost the connection with the basal lamina, significant ER-a labeling was found on the plasma membrane and this distribution was clearly apparent on D5 samples (Fig. 3D). Distribution of labeling along the PM had been changed during the inflammatory process: while in control samples the labeling showed a continuous line at the PM (Fig. 3A), it clustered at certain areas of the plasma membrane in treated cells.
Since we found no detectable differences in ER-a labeling between female and male animals, we further carried out our experiments with samples taken from male rats if not indicated otherwise.
To further clarify the presence of ER-a in mesothelial cells we measured its expression at the mRNA level by quantitative RT-PCR. Our results revealed that control mesothelial cells expressed ER-a mRNA and it showed a significant downregulation during the progression of the inflammatory process (Fig. 3E). (We found the same pattern of changes in the mRNA expression levels of ERb and G protein-coupled receptor 30, GPR30 as well (Fig. S1.).)

Estrogen receptor alpha associates with caveolin-1 both in non-treated and treated mesothelial cells
Since there are controversial data in the literature on the subcellular localization of ER-a in various cell types, we carried out double immunolabeling to detect the fine localization of ER-a as well as their possible co-localization with the coat protein caveolin-1. Our confocal microscopical results show that under inflammatory conditions ER-a colocalized with caveolin-1 both inside the cytoplasm and in the PM (Fig. 4). There were no differences in the distribution of the two markers between D3 and D5 (data not shown). However, in those mesothelial cells that were partially attached to the basal membrane, there was a remarkable colocalization of ER-a and caveolin-1 on the luminal surface of the cells (Fig. 4A). In contrast, with the disintegration of the basal membrane from the third day, ER-a appeared all over the plasma membrane in caveolin-positive membrane domains and colocalization could be detected also inside the cytoplasm (Fig. 4B). To obtain a more precise view on the localization of ER-a at the ultrastructural level, we carried out immunocytochemistry on ultrathin frozen sections of both non-treated and treated cells. Consistent with the immunofluorescence data, the same distribution of ER-a and caveolin-1 could be found on double-labeled ultrathin frozen sections of treated cells. The immunoelectron microscopic results clearly showed that at the peak time of inflammation (D3 and D5) ER-a occurred not only at the plasma membrane but appeared also inside the cytoplasm (Fig. 5A, B). Here ER-a (together with caveolin-1) was localized in forming or mature MVBs either in their limiting membrane, in association with caveolin-1 or in caveolae, in close vicinity of MVBs (Fig. 5A,  B). In untreated, control mesothelial cells ER-a labeling was accumulated along the plasma membrane on both the luminal and basolateral sides of the cells, preferentially in caveolin-positive vesicles, caveolae (Fig. 5C). ER-a labeling was also observed inside the nuclei of both untreated and treated mesothelial cells, but to a lesser extent.

Discussion
Epithelial-mesenchymal transition (EMT) is a complex mechanism that is characterized by a series of events including loss of cell-cell junctions and cell-matrix adhesion, reorganization of cytoskeleton resulting in loss of apical-basolateral polarity and assuming a mesenchymal chararacter such as spindle-shape morphology and motility. Due to its role in tumor metastasis as well as in normal embryogenesis and wound healing, the EMT is currently under intensive investigations [1]- [5].
In a previous paper we have shown that rat mesenteric mesothelial cells undergo EMT upon inflammatory stimuli induced by Freund's adjuvant treatment [26]. In the present paper we furnish additional evidence for the inflammatory response of the tissue by showing that the morphological changes at the ultrastructural level were in correlation with the expression levels of pro-inflammatory cytokines (IL 1a, 1b and IL-6) measured in mesothelial cells. This finding further supports the theory that mesothelial cells may contribute to the amplification of inflammatory responses by secreting interleukines and in this way, they are able to play an active role in peritoneal inflammatory events [34]- [36]. Proinflammatory cytokines (TNFa, IL1, IL6) are well known to induce immune and inflammatory responses by binding to their receptors. The major source of proinflammatory cytokines is the macrophages that produce plethora of these molecules including TNFa, IL-1, IL-6, Il-12, IL-15 and IL-18 [37]. Macrophages also secrete TGF-b at the site of injury that has a role in inducing EMT [1], [2], [5]. TGF-b is an important morphogenic factor initiating and maintaining EMT via different signaling routes (Smad-dependent, Smad-independent). Smadindependent signaling pathways including the activation of Erk, JNK, p38 MAP kinase cascades are considered to help and complete the process of TGF-b induced EMT. Although there are limited data about the exact mechanisms and biological consequences of these accessory signaling routes, it is apparent that TGF-b induced canonical and non-canonical pathways intersect and both are essential for effective signaling [38]- [41]. Similarly, we found significant elevation in the secreted TGF-b concentra- tion as the inflammation progressed indicating its main role to organize the EMT upon treatment in our in vivo system as well.
Earlier results from our laboratory have shown that upon adjuvant stimulation mesothelial cells are detached from the basal lamina and assume a macrophage character expressing ED1, a macrophage marker [27] and they can serve as a source of activated macrophages during inflammatory events [28]. Since macrophages are known to express ER-a [29], [30], the question arose whether treated and untreated mesothelial cells do produce ER-a as well? Our present morphological (confocal and immuno-EM) and biochemical (qRT-PCR) results show that ER-a is really  present in treated as well as in control mesothelial cells. This finding is of special interest since recently it was shown that in addition to its well known genomic role as a transcription factor, ER-a has several non-genomic functions. Nowadays it became generally accepted that a certain amount of ER-a resides in the plasma membrane and is thought to be responsible for inducing both genomic and non-genomic actions by activating different cascades, like PKC, Src kinase, MAPK and PI3K [17]- [23]. Our present light microscopic data showed that in parallel with the inflammatory events the labeling intensity of ER-a was significantly increased and could be detected in the cytoplasm, plasma membrane as well as in the nucleus. Besides the novel finding that ER-a is expressed in mesenteric mesothelial cells, the nuclear pool of the receptor was less pronounced and unchanged during the inflammatory events. This directed our focus to study further the cytoplasmic/plasma membrane pool of ER-a. In mesothelial cells, still in contact with the basal lamina, the distribution of plasma membrane ER-a pool showed a certain polarity with a predominant localization on the luminal side, while after being detached ER-a was distributed all over the plasma membrane. This redistribution of ER-a in the plasma membrane indicates loss of cell polarity and is a further proof for the transformation of a polarized epithelial cell into a non-polarized mesenchymal cell.
An interesting finding in our study was that the mRNA level of ER-a showed an inverse correlation with the labeling intensity of the protein on our immunocytochemical specimens demonstrating that on a transcriptional level ER-a was downregulated. Several studies have already described that mRNA and protein levels do not always correlate. The exact mechanism is still unclear, a possible explanation for this phenomenon can be a protein-protein interaction, the altered turnover of the molecule and posttranscriptional regulation by microRNAs [42]- [49].
A further finding in this study is that both plasma membrane ER-a and the cytosolic receptor pool were predominantly associated with caveolin-1 protein. Similar findings are known from literature showing that ER-a resides in caveolin positive raft compartments of the plasma membrane in many cells [14], [50]. Caveolin-1 is the major protein component of special highly hydrophobic membrane domains, called caveolae. Caveolae are omega-shaped, small plasma membrane invaginations that are found in most mammalian cell types. They play important role in many cellular functions including vesicular transport, endocytosis, transcytosis and are also described to be involved in different signal transduction events [51]- [55]. Collectively these data suggest that caveolae are platforms of plasma membrane where, besides other transduction proteins, ER-a is also present. The fine structural distribution of the receptor demonstrated by our immuno-electron micrographs suggests that plasma membrane ER-a is internalized upon inflammatory stimuli and is targeted to the classical endosomal pathway: towards cytoplasmic organelles, multivesicular bodies. This finding is not unusual, since endocytosis of membrane receptors and their transport along the endosomal pathway have been described in other signaling systems as well [56], [57]. Since caveolae are often considered as 'signaling platforms' [58], [59], caveola-mediated endocytosis results in a) removing signaling molecules (ERalpha, src) from the cell surface b) providing pathway for biologically important signal transducers to reach the proper cytoplasmic platforms (late endosomes, MVBs) where the microenvironment is optimal for their interaction. Considering the colocalization of ER-a with cavolin-1, it is likely that internalization is caveola-mediated. This suggestion is further supported by Christensen et al. showing that inhibition of Cav-1 by siRNA reduced membrane estrogen-receptor a levels [60]. Our further observation was that by D6 the plasma membrane of mesothelial cells devoids both clathrin-coated vesicles and caveolae (Fig. S2) indicating that internalization of signaling molecules (ERa, Src-data not shown) in caveolae is presumably important in the process of EMT. It is known that the association of caveolin and ER-a can activate MAP kinase pathways by interacting either with the signaling proteins or with each other [61]- [62]. The best studied interaction in raft domains of the plasma membrane is the induction of Erk pathway through Src/Shc/Grb2/Sos protein complex formation where these proteins physically interact with both caveolin-1 and ER-a and are activated after TGF-b stimulus [64], [65]. Taking into consideration these data, we suppose that ER-a might have a role in our system by modifying the activity of main signaling pathways via maintaining physical interaction with signaling proteins such as caveolin-1 and Src (data not shown).
There are limited data available about the localization of the elements of MAPK cascades inside the cytoplasm. The Erk pathway is well characterized and these studies illustrate the role of endosomal compartments. Either the upstream elements of these non-canonical pathways are described to occur in the plasma membrane/membrane of early endosomes or the downstream signaling elements are bound to late endosome/MVB membranes via different adaptor proteins [66]- [68]. One of these adaptor proteins (p18) has already been proved to associate with cavolin -1 indicating that upon internalization, the lipid compound of caveolae -that partially form the limiting membrane of these endosomal compartments-have importance [69]. They presumably maintain a physical platform to bind the elements of signaling proteins and in this way, they can orchestrate the spatial and temporal regulation of different signaling routes. In this paper we showed that ER-a was found either in the limiting membrane of MVBs colocalizing with caveolin-1 or in caveolae in close vicinity to late endosomal/MVB compartments. Comparing the distribution of the receptor and our EM images showing the MVB plaques, we hypothesize that the membrane of endosomal compartments/MVBs can form platforms for molecular clusters and can organize spatial and temporal distribution of signaling proteins. Although our results do not prove directly whether PM ER-a induces signaling from the plasma membrane and whether it controls accessory pathways, however, its association with caveolin-1 and Src (data not shown) strongly suggest that it might play a role in signaling events.
Recent investigations also revealed that ER-a can enhance the degradation of Smad proteins inside the nucleus via ubiquitinproteasome system and inhibit TGF-b signaling [24]. These data illustrate the role of ER-a as a non-genomic nuclear modifier of TGF-b signaling cascade.
The main aim of this study was to bring into focus the role of in vivo cell populations that can help examine the biological processes of EMT. Our work also draws attention to the role of cellular compartmentalization (caveolae and endosomes/MVBs) that can maintain platforms for possible interactions of regulatory proteins and spatially-temporally might orchestrate the signaling events. Our morphological observations and biochemical data can contribute to form a potential model whereby possible ERa-TGFb cross-talk can further be investigated in vivo. However fine details of the mechanisms and further functional evaluations are essential to clarify the exact role of ER-a in mesothelial cells under inflammatory conditions. Figure S1 The changes of mRNA expression levels of ER-b and GPR30 upon inflammatory stimuli. Similarly to ER-a, the mRNA levels of ER-b and G protein-coupled receptor 30, GPR30 showed a significant downregulation upon treatment compared to control group.