Dubravko Rendic

Jaroslav Klaudiny

Ute Stemmer

Julia Schmidt

Katharina Paschinger

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

Dubravko Rendić

Towards abolition of

Keywords: honeybee, fucosyltransferase, anti-horseradish peroxidase, N-glycan, Lewis X

come

Introduction

The glycosylation potential of insect species is increasingly being investigated not only to understand the wide range of glycotypes in nature, but in order to determine the origin of immunogenic and allergenic cross-reactive carbohydrate determinants and to refine the use of insect-based expression systems in the production of pharmaceutically-relevant glycoproteins. In both these cases, the non-mammalian features of insect N-glycans are relevant: whereas many mammalian glycoproteins carry sialic acids on so-called 'complex' N-glycans, insect glycoproteins have distinct features which are 'foreign' to mammalian immune systems (Figures 1A and1B). However, the 'humanisation' of glycans of insect cell expression systems may aid the manufacture of glycoprotein drugs, such as erythropoietin or clotting factors, which have otherwise to be produced at considerable cost from mammalian cell lines or with the risk of using natural sources contaminated with viruses or prions. From the biotechnology perspective, the complete lack (or very low levels) of terminal sialylation [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF][START_REF] Butters | Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunciamycin[END_REF][START_REF] Roth | Occurrence of sialic acids in Drosophila melanogaster[END_REF][START_REF] Hooker | Constraints on the transport and glycosylation of recombinant IFN-γ in Chinese hamster ovary and insect cells[END_REF][START_REF] Lopez | Oglycosylation potential of lepidopteran insect cell lines[END_REF][START_REF] North | Glycomic studies of Drosophila melanogaster embryos[END_REF] and the processing of the oligosaccharides to 'paucimannosidic' forms (such as structures in Figure 1B) are a particular problem when using insect cell lines [START_REF] Wagner | Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human β1,2-N-acetylglucosaminyltransferase I[END_REF][START_REF] Donaldson | Use of mannosamine for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells[END_REF][START_REF] Hollister | Engineering the protein N-glycosylation pathway in insect cells for production of biantennary complex N-glycans[END_REF][START_REF] Léonard | The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing[END_REF], since such glycoforms may be cleared quickly from the mammalian circulation by lectin-dependent mechanisms. Moreover, the presence of α1,3-fucose bound to the proximal Nacetylglucosamine of an N-glycan (i.e., core α1,3-fucose; see Figures 1B and1C), as also found in plants including on pollen and food allergens, is of concern for both allergologists and biotechnologists because of its immunogenicity [START_REF] Tretter | Fucose α1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals[END_REF][START_REF] Paschinger | Definition of immunogenic carbohydrate epitopes[END_REF]. Indeed, core α1,3-fucose constitutes a major epitope recognised by antisera raised in rabbits against insect and plant glycoproteins, such as bee venom or horseradish peroxidase [START_REF] Prenner | The antigenicity of the carbohydrate moiety of an insect glycoprotein, honey-bee (Apis mellifera) venom phospholipase A 2 . The role of α1,3fucosylation of the asparagine-bound N-acetylglucosamine[END_REF][START_REF] Kurosaka | The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum[END_REF]. Furthermore, in one study, the sera of about 25% of patients with bee venom allergy were found to contain IgE cross-reacting with plant N-glycans, an observation explained by the presence of core α1,3fucose in both plants and insects [START_REF] Tretter | Fucose α1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals[END_REF]. Similar findings have been reported for patients allergic to fire ant [START_REF] Hoffman | Sol i 1, the phospholipase allergen of imported fire ant venom[END_REF] and wasp venoms [START_REF] Hemmer | Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy[END_REF].

Detailed analysis of the N-glycans of two components of bee venom, phospholipase A 2 and hyaluronidase (designated respectively as allergens Api m 1 and Api m 2), showed that these glycoproteins possess a number of non-mammalian features (see Figure 1D for the most complex structure observed). Besides the core α1,3-fucosylation mentioned above, some glycoforms of both these proteins carry a Lewis-type fucosylated LacdiNAc structure [17,18], which resembles the Lewis X structure (Le x ; Figure 1E), except that a GalNAc is present rather than a galactose. Fucosylated LacdiNAc is also present in parasitic trematode and nematode worms (particularly Schisotsoma spp. and Haemonchus contortus) and is a major epitope for antibodies from infected individuals or animals [19,[START_REF] Vervelde | Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen[END_REF]. In humans, Lewistype epitopes containing galactose constitute a set of carbohydrate histo-blood group antigens. These moieties are often modified by sialic acids and sulphate groups, and as such play numerous roles including ones in inflammation and cancer [START_REF] Becker | Fucose: biosynthesis and biological function in mammals[END_REF][22][23].

Based on the aforementioned data, we hypothesised that at least two α1,3fucosyltransferases would be encoded by the honeybee genome. To date, all enzymes required for addition of α1,3-linked fucose residues to glycans have been found to be members of one enzyme family, CAZy glycosyltransferase family 10 [START_REF] Coutinho | An Evolving Hierarchical Family Classification for Glycosyltransferases[END_REF], which includes both core α1,3-fucosyltransferases from plants, nematodes and insects as well as enzymes from bacteria, plants and enzymes of mammals capable of generating Lewis blood group antigens. Previously, in insects, the native enzyme activity responsible for the acquisition of the core α1,3-fucose epitope has been described in bee venom glands [START_REF] Staudacher | GDP-fucose: β-N-acetylglucosamine (Fuc to (Fucα1→6GlcNAc)-Asnpeptide) α1→3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparagine-bound N-acetylglucosamine[END_REF] and in a cell line [26]. However, the only data previously obtained on recombinant insect fucosyltransferases pertains to Drosophila melanogaster: its genome encodes four α1,3-fucosyltransferase homologues (DmFucTA, DmFucTB, DmFucTC and DmFucTD). Of these only DmFucTA, a core α1,3-fucosyltransferase with orthologues in all other insect genomes examined, has a defined enzymatic activity [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF][START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF]. On the other hand, no Lewis-type enzyme is expected to be encoded by the fruitfly genome since no relevant epitopes have been described in Drosophila to date; thus, it is to be assumed that any honeybee enzyme with a Lewis-type fucosyltransferase may not be so closely related to the three 'orphan' fucosyltransferases (FucTB, FucTC and FucTD) from the fruitfly.

To test the hypothesis that a honeybee fucosyltransferase homologue least akin to DmFucTA may indeed be a Lewis-type enzyme able to fucosylate LacdiNAc, whereas putative orthologues of DmFucTA are solely core-type enzymes, we searched the draft honeybee genome and EST databases for α1,3-fucosyltransferase homologues. Of three fucosyltransferase homologues identified, two were indeed expressed as active proteins in both insect cells and yeast and were subject to a variety of biochemical assays in order to define their enzymatic function.

Experimental Procedures

Cloning of fucosyltransferase cDNAs from the honeybee

Expression of honeybee putative fucosyltransferases

Constructs in the pIZT-V5/His and pPICZα-type vectors were transformed respectively into insect Sf9 (Spodoptera frugiperda; fall armyworm) and Pichia pastoris GS115 cells as previously described [START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF][START_REF] Mucha | The Drosophila melanogaster homologue of the humna histoblood group P k gene encodes a glycolipid-modifying α1,4-N-acetylgalactosaminyltransferase[END_REF]. In Pichia pastoris the recombinant enzymes were expressed at 16 o C. Various strategies, as described below, were used to verify enzyme activities. Briefly, core α1,3-fucosyltransferase activity was tested either by Western blotting of transfected Sf9 cells using rabbit anti-horseradish peroxidase (anti-HRP) or in vitro using various dansyl-and dabsyl-glycopeptides as substrates as used in previous studies [START_REF] Mucha | The Drosophila melanogaster homologue of the humna histoblood group P k gene encodes a glycolipid-modifying α1,4-N-acetylgalactosaminyltransferase[END_REF]29].

The Lewis-type fucosyltransferase was also assayed with dabsyl-glycopeptides as well as with pyridylaminated lacto-N-neo-tetraose (Galβ1,4GlcNAcβ1,3Galβ1,4Glc-PA) and lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc-PA). Where stated, Pichia supernatants were concentrated 10-fold using UltraFree centrifugal concentration devices (Millipore, MWCO 30000).

MALDI-TOF-MS fucosyltransferase assay

For analysis by MALDI-TOF-MS, the activity of recombinant α1,3fucosyltransferases from concentrated Pichia supernatants was measured using 40 mM MES buffer, pH 6.5, 10 mM MnCl 2 , 1 mM GDP-fucose and 80 µM of each of dabsyl-MM, -GnGn, -GalGal and -βGNβGN glycopeptides derived from bovine fibrin, and possessing the common peptide sequence GENR (see Figure 2 for oligosaccharide structures and Ref. 28).

Reaction tubes were incubated at 30°C and analyzed after 12 h. Following incubation, 0.5 µl of a 1:10 dilution of the reaction mixture was mixed with 0.5 µl of 1% α-cyano-4hydroxycinnaminic acid (Fluka; in 70% acetonitrile) on a MALDI plate. An increase in glycopeptide m/z by 146.1 Da indicated the transfer of one fucose residue.

RP-HPLC based fucosyltransferase assays

For analysis by HPLC, the activity of concentrated supernatants of Pichia expressing honeybee FucTA was measured using 40 mM AMPD and MES buffer, pH 6.5-9.5 (MES buffer, pH 7.0 for temperature and cation dependency), 10 mM MnCl 2 , 1 mM GDP-fucose, and 25 µM dansylated GnGnF 6 -glycopeptide derived from human IgG, with NS as the peptide sequence (see Figure 2 for oligosaccharide structure). Reaction tubes were incubated at 23 °C and analyzed after 2 h. Following incubation an aliquot of the reaction was analysed on a reverse-phase HPLC system (Hypersil ODS) under isocratic conditions (9.5% acetonitrile, 0.045% trifluoroacetic acid).

The activity of unconcentrated supernatants of Pichia expressing honeybee FucTC was measured using 50 mM AMPD and MES buffer pH 6.5-9.5 (MES buffer, pH 7.5 for temperature and cation dependency), 100 µg/µl bovine serum albumin, 12.5 mM MnCl 2 , 1.25 mM GDP-fucose and 125 µM pyridylaminated lacto-N-neo-tetraose. Reaction tubes were incubated at 23 °C and analyzed after 2 h. Following incubation, an aliquot of the reaction was analysed on a reverse-phase HPLC system (Nucleosil C18) under isocratic conditions (0.094 M ammonium-formate, pH 4.0, 1.8% methanol).

Western blotting

GnGn, GalGal and βGNβGN isoforms of human apo-transferrin were prepared by enzymatic remodelling using Clostridium perfringens neuraminidase (yielding GalGaltransferrin), Aspergillus oryzae β-galactosidase (yielding GnGn-from GalGal-transferrin)

and bovine β1,4-galactosyltransferase I (yielding βGNβGNfrom GnGn-transferrin since the enzyme also utilises UDP-GalNAc as a sugar donor [START_REF] Mucha | The Drosophila melanogaster homologue of the humna histoblood group P k gene encodes a glycolipid-modifying α1,4-N-acetylgalactosaminyltransferase[END_REF][START_REF] Palcic | Flexibility in the donor substrate specificity of β 1,4galactosyltransferase: application in the synthesis of complex carbohydrates[END_REF]). Recombinant honeybee FucTA (concentrated) and FucTC (unconcentrated) were incubated with 20 µg of the transferrin neoglycoforms in 40 µM MES, pH 7.0, 10 mM MnCl 2 , 1 mM GDP-Fuc and 1 mM PMSF in a total volume of 5 µl. The reactions were performed at 23 o C for 48h. One-fifth of each incubation was mixed with an equal volume of 2× Laemmli buffer and analysed by Western blotting using 1:10000 dilutions of either rabbit anti-horseradish peroxidase (anti-HRP) or rat anti-Le x (L5) as primary antibodies and 1:2000 dilutions of the relevant secondary alkaline phosphatase-conjugated antibodies followed by development using SigmaFAST BCIP/NBT solution.

Sf9 cells were maintained, transfected and lysed as previously described [START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF]. Briefly, pIZT/V5-His vectors carrying complete ORFs of all three Apis mellifera α1,3fucosyltransferase homologues with the native stop codon were used to transfect Sf9 cells (maintained in IPL41 medium supplemented with 3% FCS) using Cellfectin reagent Western blotting using a 1:20000 dilution of rabbit anti-HRP as primary antibody and a 1:2000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) as secondary antibody followed by development using SigmaFAST BCIP/NBT solution. Prior to blocking, the nitrocellulose sheets were reversibly stained with 0.5% Ponceau S (in 1% acetic acid) to verify that equal amounts of proteins were present in every lane.

RT-PCR analyses

Honeybees (Apis mellifera carnica) were obtained from one colony at a private apiary located near Bratislava (Slovak Republic). Nurses were collected on a comb taken from the hive in the region containing cells with 1-3 day-old larvae. Foragers were caught in an insect The primer concentration in the reaction was 0.8 µM. The concentration of Mg 2+ was 1.5 mM except for the amplification of FucTB, where the optimal concentration was 1.2 mM.

Reverse transcription was performed at 48°C for 1 h. PCR after the initial denaturation for 2 min at 94°C was performed for 30, 34 or 50 cycles (30s at 94°C, 60s at 54°C and 150s at 68°C) followed by 7 min at 68°C. PCR products were analysed on 1% agarose gels.

Results

Cloning of fucosyltransferase cDNAs from the honeybee

The first target was to identify which gene is responsible in the honeybee for the occurrence of core α1,3-fucose on bee venom glycoproteins; it seemed reasonable that the shown by the finding that a Drosophila FucTA probe could recognise the honeybee FucTA PCR product, but not that of fruitfly FucTB, at low stringency (2× SSC, 0.1 % SDS, 1 hr, 55 °C; data not shown). The determined honeybee FucTA cDNA sequence was identical to that later released for the predicted mRNA XM_392653 (based on honeybee Contig1747); a comparison of the cDNA and genomic sequences also showed that the honeybee FucTA is encoded by three exons.

Considering the presence of fucosylated LacdiNAc on bee venom glycoproteins, we also sought a second fucosyltransferase gene. Indeed, when using the fruitfly FucTC sequence to search the now available honeybee genome, another predicted fucosyltransferase was found (XM_394138; honeybee Contig655); the confirmed cDNA sequence encodes a protein of 399 residues (M r 47 kDa), which we designate as FucTC. Furthermore, using the amino acid sequence of a Glossina morsitans putative fucosyltransferase (Genbank entry AJ582622), another potential homologous sequence was found on honeybee Contig4553; although this was not a predicted mRNA, a corresponding cDNA designated FucTB was then successfully cloned. This sequence encodes a protein of 431 amino acids (M r 51 kDa), which instead of the DYI/VTEK motif typical of animal α1,3-fucosyltransferases has an EYVTEK sequence. The three honeybee fucosyltransferases display between 28 and 34% identity to each other over approximately 300 residues (Figure 3A).

Phylogenetic analysis (Figure 3B) groups the honeybee and fruitfly FucTAs together, whereas those designated as FucTB homologues appear to form two distinct groups: one group is more related to the human Lewis-type fucosyltransferases III-VII and IX, while the other is closer to the mammalian fucosyltransferase homologues X and XI. The honeybee FucTC appears to be closely related to a number of insect sequences; however, a branching into two distinct FucTC groups cannot be excluded. Indeed, the occurrence of two FucTC groups would be in agreement with the apparent inability of fruitfly FucTC to utilise various N-glycan substrates [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF][START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF], whereas, as shown below, honeybee FucTC does accept such glycans.

Expression of honeybee fucosyltransferases in Pichia

Constructs encoding FLAG-tagged soluble forms of the three honeybee fucosyltransferase homologues were prepared for expression in the yeast Pichia pastoris.

Culture supernatants were collected and concentrated prior to assaying specific fucosyltransferase activities with various dabsylated N-glycopeptides (MM, GnGn, GalGal and βGNβGN; see Figure 2 for structures). MM and GnGn were used as known core fucosyltransferase substrates, whereas GalGal and βGNβGN are substrates for Lewis-type enzymes [31]. As shown in Figure 4, Pichia transformed with the FucTA vector converted GnGn to a fucosylated form, whereas that transformed with FucTC vector converted GalGal and βGNβGN to difucosylated forms. Upon longer incubation (36h), the honeybee FucTA expressed in Pichia also converted GalGal and βGNβGN to monofucosylated forms (data not shown). FucTB-transformed yeast showed no obvious activity in the initial screening, thus all further experiments were performed on FucTA and FucTC.

FucTA activity was also measured using HPLC-based assay with the fluorescentlylabelled dansylated-GnGnF 6 glycopeptide as the substrate. In agreement with previous studies on core α1,3-fucosyltransferases [START_REF] Roitinger | HPLC method for the determination of Fuc to Asn-linked GlcNAc fucosyltransferases[END_REF], the addition of a fucose by FucTA lead to reduction of the substrate glycan retention time (Figure 5). Since FucTC appeared to be a Lewis-type enzyme, we performed HPLC-based assays using pyridylaminated forms of the tetrasaccharides lacto-N-neo-tetraose and lacto-N-tetraose as substrates, since these have been successfully used with Lewis-type fucosyltransferases forming, respectively, α1,3 and α1,4fucosyl linkages. Previous studies with a plant Lewis-type α1,3/4-fucosyltransferase (tomato FucTC) showed that fucosylation of these substrates results in a shift to earlier elution times [33]. In the present study with honeybee FucTC, only in the case of lacto-N-neo-tetraose was there an alteration in the retention time, resulting from formation of lacto-N-fucopentaose III (Figure 5); no such shift was observed for yeast transformed with FucTA or FucTB nor when lacto-N-tetraose was incubated with FucTC (data not shown). These data, therefore, show that the enzyme solely acts as an α1,3-fucosyltransferase. The determined activity of FucTC towards both LacNAc and LacdiNAc-containing substrates is comparable to data showing that the mammalian fucosyltransferases III, IV and IX have varying levels of activity in vitro towards both LacNAc (in order to generate Le x ) and LacdiNAc, whereas FucT VII displays no activity towards LacdiNAc [START_REF] Cailleau-Thomas | FUT4 and FUT9 genes are expressed early in human embryogenesis[END_REF].

Enzymatic properties of honeybee FucTA and FucTC

The basic enzymological parameters of FucTA and FucTC were investigated using the aforementioned HPLC-based assays. Honeybee FucTA is strongly activated by Mn 2+ as compared to no-cation controls, a property common to many glycosyltransferases. On the other hand, EDTA, as well as Cu 2+ and Zn 2+ ions, completely abolish this enzyme's activity.

The enzyme shows maximal activity in mildly alkaline conditions (pH 7.5-9.0) at room temperature. In contrast to FucTA, FucTC is not inactivated by EDTA, although the enzyme seems to prefer Mn 2+ and Mg 2+ over other cations. The temperature and pH optima for the FucTC appear to be very broad; an enzyme shows 90% of maximal activity at 4 o C and over 40% of maximal activity at 50 o C (Figure 6).

The honeybee FucTA and FucTC can utilise protein substrates

The assays employed to measure the activity of both core and Lewis-like α1,3fucosyltransferase utilise substrates containing predominantly carbohydrates, with little or no peptide backbone as compared to "real" glycoproteins. Furthermore, these assays primarily indicate transfer of fucose but do not give information about the epitopes formed. To address this major difference between utilised substrates and the ones present in vivo, we made use of the human glycoprotein apo-transferrin whose glycans were modified to be potential substrates for the two recombinant enzymes. After incubation with GDP-fucose and Pichia culture supernatants containing either FucTA or FucTC, the glycoprotein preparations were separated on a standard SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with anti-HRP or anti-Le x antibodies. Whereas anti-HRP is known to bind core α1,3-linked fucose as found on many plant and insect glycoproteins [START_REF] Wilson | Core α1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts[END_REF], the L5 monoclonal antibody is apparently the only antibody capable of recognising the Le x epitope when attached to Nglycans [36]. The transferrin neoglycoforms carrying N-glycans predominantly terminating with β1,2-GlcNAc (i.e., GnGn-), β1,4-Gal (i.e., GalGal-) and β1,4-GalNAc (i.e., βGNβGNtransferrin) residues incubated with FucTA were recognised by anti-HRP antibodies demonstrating that transfer of a fucose resulted in generation of the same core α1,3-fucose epitope as found on, e.g., horseradish peroxidase and bee venom phospholipase. On the other hand, only GalGal-transferrin incubated with FucTC was recognised by antibodies directed against the Le x structure, thus strongly indicating that the fucose residues were attached in α1,3-linkage to the antennal N-acetylglucosamine residues (Figure 7A). Even though FucTC can transfer fucose to βGNβGN, the specificity of the L5 antibody is obviously absolute for Le x and therefore this antibody does not recognise fucosylated LacdiNAc.

Analysis of honeybee fucosyltransferases activity in vivo

In previous studies, we have used insect cells as hosts for core α1,3- FucTC showed no difference as compared to the empty vector control corroborating the other data suggesting that they are not core fucosyltransferases. Furthermore, in vitro assays also showed that only the FucTA-transfected cells had an activity capable of transferring a fucose to GnGnF 6 , thereby generating a difucosylated structure, above the otherwise very low background levels displayed by control transfectants (data not shown). The overall conclusion is that, as judged by data indicating that this enzyme can form the anti-HRP epitope in vitro and in vivo, the fucose transferred by FucTA is α1,3-linked to the proximal GlcNAc of N-glycans.

Expression of fucosyltransferases in different honeybee tissues

To appraise whether the tissue-specific expression of α1,3-fucosyltransferase homologues in Apis mellifera is compatible with the synthesis of the previously observed Nglycans containing both core and antennal α1,3-linked fucose [17], total RNA from several honeybee tissues was prepared. Following RNA normalisation against actin transcripts, expression pattern of the fucosyltransferase homologues in honeybees was estimated by RT-PCR using gene specific primers. As expected, the expression panel indicates that there is a very strong presence of FucTA transcripts in venom glands, followed by strong expression of FucTA in bee brain and weaker expression in the other analysed samples (Figure 8). FucTC, on the other hand, appears to be expressed more equally in various samples, with an apparently higher expression rate in venom and hypopharyngeal glands. The reproducible appearance of the triplet band in FucTC RT-PCR reactions for most tissues could indicate alternative splicing of the FucTC transcripts. Indeed TA cloning and subsequent sequencing confirmed the existence of two alternatively spliced forms of FucTC; one corresponded to that expressed in Pichia, whereas the second form is 90 nucleotides shorter and has a premature stop codon. Finally, the third α1,3-fucosyltransferase homologue, FucTB, appears to be weakly expressed in venom glands, foragers' brain tissue and in larvae; it should be noted that it was necessary to increase the number of RT-PCR cycles to 50 and use increased amounts of RNA in order to detect FucTB transcripts in honeybee RNA preparations.

Overall, the expression panel data indicating the presence of both the core-modifying FucTA and the Lewis-like FucTC in venom glands is in accordance with the previous studies indicating the presence of both core and antennal α1,3-fucosylated N-glycan structures on bee venom glycoproteins.

Discussion

The honeybee Apis mellifera is an object of scientific study for a number of reasons; in particular, the search for genetic clues as to its social behaviour has led to the recent sequencing of its genome and the examination of bee brain EST sequences [37], but also the anaphylaxis induced in some individuals, as an allergenic reaction to bee stings, is a focus of allergological interest [START_REF] Tretter | Fucose α1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals[END_REF]. The present study has its origins in previous enzymological and structural analyses related to bee venom glycoproteins [17], which then showed, for the first time, the presence of both core α1,3-fucose and α1,3-fucosylated LacdiNAc structures on an insect glycoprotein. Thus, we expected to find at least two genes in the honeybee encoding enzymes able to synthesise these structures.

In actual fact, the Apis mellifera genome encodes three α1,3-fucosyltransferase homologues which include the consensus sequence common to all α1,3-fucosyltransferases [START_REF] Breton | Conserved structural features in eukaryotic and prokaryotic fucosyltransferases[END_REF]39]. As indicated by the predicted amino acid sequences, one of the honeybee sequences (FucTA) is phylogenetically close to Drosophila and other proven or putative N-glycan core α1,3-fucosyltransferases (Figure 3B). Indeed, when expressed in Pichia pastoris and insect cells, this honeybee sequence encodes an active core α1,3-fucosyltransferase with a substrate specificity similar to that of the recombinant Drosophila FucTA [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF], the native honeybee venom gland and Mamestra brassicae MB-0503 (cabbage moth cell line) core α1,3fucosyltransferases [START_REF] Staudacher | GDP-fucose: β-N-acetylglucosamine (Fuc to (Fucα1→6GlcNAc)-Asnpeptide) α1→3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparagine-bound N-acetylglucosamine[END_REF]26] and various plant N-glycan core α1,3-fucosyltransferases [40][START_REF] Leiter | Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase from mung beans[END_REF][START_REF] Wilson | Cloning and expression of α1,3-fucosyltransferase homologues from Arabidopsis thaliana[END_REF]. The observed transfer of fucose to GalGal, in addition to GnGn, has also been found with the fruitfly FucTA [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF]. On the other hand, no transfer was seen towards MM by either insect FucTA, which contrasts to the preference of the Caenorhabditis FUT-1 for this substrate [29]. This suggests that the substrate specificity of invertebrate core α1,3fucosyltransferases (i.e., FucTA and FUT-1) is somewhat more variable and flexible than that of invertebrate core α1,6-fucosyltransferases which require a terminal unsubstituted GlcNAc residue [31].

The second homologue found in this study (FucTC) resembles a number of other related sequences from various insects, including the Drosophila FucTC sequence. Unlike DmFucTC [START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF], the honeybee FucTC encodes an active Lewis α1,3-fucosyltransferase and is indeed the first such enzyme to be characterised from an insect; it is very likely that this enzyme is involved in synthesis of fucosylated LacdiNAc N-glycans found on bee venom glycoproteins [17]. It appears that, the FucTC homologues form two distinct subgroups; the honeybee FucTC seems to be more related to Bombyx mori and Tribolium castaneum sequences, whereas the Drosophila FucTC is more similar to Anopheles gambiae, Aedes aegypti and Glossina morsitans sequences. However, it remains to be determined whether this genetic 'split' is generally reflected in the presence or absence of fucosylated LacdiNAc.

Apart from demonstrating that both honeybee FucTA and FucTC can act on glycoproteins and that FucTA is able to confer fucose-dependent anti-HRP staining in an in vivo system, albeit heterologous, we sought to strengthen the link between the honeybee α1,3-fucosyltransferase homologues and fucosylated N-glycan structures found on bee venom glycoproteins [17,18] by performing expression studies of all three honeybee α1,3fucosyltransferase homologues in different bee tissues. According to the RT-PCR expression panel, FucTA appears to be strongly expressed in venom glands, a result that correlates with the presence of the core α1,3-linked fucose on the N-glycans of bee venom glycoproteins.

The occurrence of FucTA transcripts in bee brain tissue is also in agreement with expression of DmFucTA in Drosophila heads and in a Drosophila neuronal cell line [START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF] and in the use of anti-HRP as a neuronal marker in insects [43]. The honeybee FucTA also appears to be expressed in hypopharyngeal glands that produce the royal jelly proteins in honeybee nurses [START_REF] Knecht | Patterns of larval food production by hypopharyngeal glands in adult worker honey bees[END_REF][START_REF] Ohashi | Change in the mode of gene expression of the hypopharyngeal gland cells with an age-dependent role change of the worker honeybee Apis mellifera L[END_REF][START_REF] Schmitzova | A family of major royal jelly proteins of the honeybee Apis mellifera L[END_REF] and carbohydrate-metabolising enzymes after the bees adopt the forager role [START_REF] Kubo | Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role[END_REF][START_REF] Ohashi | Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L.)[END_REF][START_REF] Simpson | Invertase in the hypopharyngeal glands of the honeybee[END_REF].

This result contrasts the apparent lack of fucosylated N-glycan structures on major royal jelly glycoproteins [START_REF] Kimura | Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans[END_REF]. The explanation could be that transcripts detected in muscles and the hypopharyngeal gland might originate from neighbouring neuronal tissue which is expected to express the FucTA. On the other hand, antibodies detecting N-glycan core α1,3-linked fucose appear also to bind other tissues in Drosophila (e.g., the garland gland) [43, [START_REF] Snow | Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embyros[END_REF][START_REF] Seppo | Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells[END_REF],

indicating that α1,3-linked fucosylated N-glycan structures might occur in insect tissues other than, as generally accepted, neurons [START_REF] Fabini | Identification of core α1,3fucosylated glycans and the requisite fucosyltransferase in Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope[END_REF][START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF] and venom glands [START_REF] Tretter | Fucose α1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals[END_REF]17,18].

Honeybee FucTC appears to be expressed in all tissues analysed and the reproducible appearance of the triplet band in FucTC RT-PCR reactions (where the largest band is of the expected size) for most tissues could indicate alternative splicing of transcripts of this gene.

The higher expression rate of FucTC in venom glands correlates well with the occurrence of LacdiNAc structures present on bee venom glycoproteins. However, even though honeybee FucTC is expressed to a high degree in the hypopharyngeal glands, the N-glycans of major royal jelly glycoproteins appear, as mentioned above, not to be fucosylated. It was also observed that the expression of FucTC is similar in both nurse's and forager's glands that produce different proteins and show morphological and ultrastructural differences [START_REF] Deseyn | Age-dependent morphology and ultrastructure of the hypopharyngeal gland of Apis mellifera workers (Hymenoptera, Apidae)[END_REF]. On the other hand, the dual in vitro activities of FucTC, towards both LacNAc and LacdiNAc structures, may not have relevance in vivo, since only fucosylated forms of the latter have been previously described on honeybee glycoproteins. It is indeed probable that the honeybee only expresses a β1,4-N-acetylgalactosaminyltransferase, akin to those previously described from Trichoplusia ni [54] and Drosophila melanogaster [START_REF] Haines | Functional analysis of Drosophila β1,4-Nacetlygalactosaminyltransferases[END_REF], and lacks a β1,4galactosyltransferase of the type found in mammals. Therefore, presumably only the substrate with terminal GalNAc, required to generate a fucosylated LacdiNAc, is present in honeybees in vivo.

The expression panel suggests that honeybee FucTB is very weakly expressed in larvae, venom glands and foragers' brain tissue but not at all in nurses' brain tissue; indeed, its expression is several orders of magnitude lower than that of honeybee FucTA and FucTC.

Interestingly, honeybee FucTB appears to be phylogenetically closer to known N-glycan core α1,3-fucosyltransferases like Drosophila FucTA and honeybee FucTA than to the Drosophila FucTB fucosyltransferase homologue and the putative human and murine fucosyltransferases X and XI. However, extensive attempts with both fruitfly and honeybee FucTB homologues to demonstrate an activity towards potential substrates (MM, GnGn, GalGal, βGNβGN, lacto-N-neo-tetraose and lacto-N-tetraose) were not successful. This, in conjunction with the fact that we were able to account for the major fucosylated structures present in N-glycans on bee venom and fruitfly glycoproteins, still leaves an open question as regards the in vivo function of FucTB proteins. However, any results with an insect FucTB might prove valuable for the understanding of the function of the homologous mammalian FucT X and XI, for which also, to date, no activity has been detected [START_REF] Baboval | Comparison of human and mouse Fuc-TX and Fuc-TXI genes, and expression studies in the mouse[END_REF]. Indeed, it may be that the assumption, that standard 'glycoprotein' substrates are applicable, is false.

In conclusion, we have identified a core α1,3-fucosyltransferase, from an insect other than Drosophila, as well as the first insect Lewis-type fucosyltransferase. These enzymes account for the N-glycan α1,3-fucosylation events previously hypothesised on the basis of Nglycan analysis of honeybee glycoproteins and both generate products associated with immune responses to glycans from invertebrates [START_REF] Tretter | Fucose α1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals[END_REF][START_REF] Prenner | The antigenicity of the carbohydrate moiety of an insect glycoprotein, honey-bee (Apis mellifera) venom phospholipase A 2 . The role of α1,3fucosylation of the asparagine-bound N-acetylglucosamine[END_REF]19,[START_REF] Vervelde | Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen[END_REF]. Despite being both members of the same enzyme family, honeybee FucTA and FucTC have different substrate specificities; indeed, as with other members of CAZy glycosyltransferase family 10, the only feature in common is the transfer of fucose, from GDP-Fuc, to GlcNAc residues. The wider significance of our findings is, however, in connection to those commonly-used baculovirus hosts such as Trichoplusia ni (HiFive ® ) and Sf21 cells [START_REF] Hsu | Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells[END_REF]58] known to express core α1,3fucosylated N-glycans, but for which we lack relevant DNA sequence information. Indeed, of the various non-Drosophila insect species for which such data is currently publicly-available, only Bombyx mori is of direct biotechnological importance with BmN cells sometimes being used for baculovirus-based expression. However, the relatively-high homology between the fruitfly and honeybee FucTA core α1,3-fucosyltransferase sequences and the observed ability of these sequences to cross-hybridise may be useful in screening cDNA libraries from baculovirus-host species for which no genomic data is currently available. Identification of the core α1,3-fucosyltransferases from such cell lines would pave the way for knockingout/down the corresponding gene, thus resulting in abolition/reduction of the expression of the immunogenic core α1,3-fucose moiety. This approach would complement other efforts to re-engineer insect cell lines [START_REF] Hollister | Engineering the protein N-glycosylation pathway in insect cells for production of biantennary complex N-glycans[END_REF][START_REF] Hill | Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT-1 cells cultured in serum-free medium[END_REF] in order to generate 'human-like' glycans on proteins of pharmaceutical interest. galactose residues as white circles, glucose residues as black circles, N-acetylglucosamines as black squares, N-acetylgalactosamines as white squares and fucose residues as grey triangles (as shown in Figure 2). Structures appearing due to enzymatic activity are underlined. Peaks resulting from laser-induced cleavage of a part of the dabsyl group are labelled with an asterisk. the presence or absence of GDP-Fuc. The FucTC was also tested using lacto-N-tetraose but did not display any activity towards that substrate (data not shown).

  Male honeybee larvae were collected from a hive in Schiltern near Krems in Lower Austria and cDNA was prepared by Trizol (Invitrogen) extraction and subsequent reverse transcription (Superscript III, Invitrogen) with oligo(dT) 18 . A fragment with a partial reading frame of the FucTA homologue was amplified using the primers BBFT3 (GAGATATGGCACCGTATGC) and BBFT2/XbaI (GCTCTAGACCTAGGCAGAAAGTTTTTC) with Expand polymerase mix (Roche) and then cloned into the pGEM-T vector (Promega). The full length form was reconstructed by ligating a BstAPI/KpnI fragment from the pGEM-T/FucTA plasmid into the corresponding sites of the EST clone (GenBank BI503295, clone ID BB170010A20D04) in pT7T3-Pac encoding only the 5´-and the 3´-ends of honeybee FucTA. This reconstructed form was used as a template for PCR using KOD polymerase (Novagen) with the primers BBFT1/NotI (ATAAGAATGCGGCCGCATGGGTCTGCCGCG) and BBFT2/XbaI; the resulting fragment was then digested with NotI and XbaI and ligated into the pIZT-V5/His vector (Invitrogen). The complete FucTB and FucTC open reading frames were prepared by PCR using, respectively, the primers BFTB1/KpnI (in combination with Expand polymerase mix and the fragments were ligated also into the pIZT-V5/His vector. Partial reading frames corresponding to the catalytic and stem regions of FucTA, FucTB and FucTC were isolated using the primers BBFT3/PstI (or BFTC3/PstI (AAACTGCAGGTACCCCTCGACATATAATAC) with BFTC2/XbaI and ligated into a form of the pPICZαB vector (Invitrogen) modified to encode a FLAG-tag.

(

  Invitrogen) following the manufacturer's protocol for insect cells. A small aliquot of the cells was analysed by confocal laser scanning microscopy with a UV-light source to confirm the presence of GFP fluorescence within the cells as indication of a successful transfection. Cells were collected 48h post-transfection, washed once with PBS and stored at -80 o C. Cells were lysed using 50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100 supplemented with Complete-mini protease inhibitor cocktail without EDTA (Roche) or His-tag protease inhibitor cocktail (Sigma) for 10 minutes at 23 o C and 20 minutes on ice followed by removal of insoluble material by centrifugation (14100g, 4 o C, 30 min). Different volumes of the buffer were used to normalise the number of cells per volume (to 1.25 × 10 4 cells/µl). Cell lysates (2.5 µl per lane) were mixed with 2× Laemmli loading buffer and analysed by

  net while trying to fly into or out of the hive. After collection, tissues and glands were dissected from distinct numbers of honeybees [thorax muscle (2 individuals), brains (10 individuals), hypopharyngeal glands (12 individuals) and venom glands (17 individuals)] into RNAlater solution and stored at -20° C. Total RNA from honeybee tissues and glands was prepared with SV Total RNA isolation System (Promega) after Ultra-Turrax homogenization in a denaturing solution. The quality and the spectrophotometrically-estimated concentration of the preparations were tested by agarose gel electrophoresis. RT-PCR assays were performed with the one-tube, two-enzyme Access RT-PCR System (Promega) using 40-160 ng (FucTA, FucTC, actin) or 100-400 ng (FucTB) of RNAs in a volume of 25 µl. The primers used in the RT-PCRs had the same Tm for the specific sequences (58°C) and also worked with the same efficiencies in PCR using plasmid DNAs containing the corresponding FucT cDNAs. Primers were designed so that products would span at least one intron/exon boundary to ensure that cDNA amplicons could be distinguished from genomic DNA amplicons. The following primers were used: FucTA: BBFT3/PstI and BBFT2; FucTB: BFTB/5 (ATAGAACATCGAAAGCAAACG) and BFTB2/XbaI; FucTC: BFTC3/PstI and BFTC2/XbaI; Actin: Actinfor (MTCCGGIATGTGCAARGCCG) and Actinrev (CCTGCTCRAAGTCMAGIGC).

  encoded protein would display homology to other core α1,3-fucosyltransferases, including the known Drosophila melanogaster FucTA. During initial searches of the EST databanks in 2001, two partial bee brain cDNA sequences from the honeybee with homology to Drosophila FucTA were noted. The two relevant clones were ordered, but both were found to contain DNA encoding protein sequences with homology to only the N-and C-terminal regions of the fruitfly FucTA. These EST DNA sequences were then used to design specific primers in order to clone the full ORF as well as an ORF fragment encoding a form lacking the putative cytosolic and transmembrane regions. Only the latter fragment was actually isolated from honeybee cDNA, but due to the presence of a BstAPI site in the cDNA sequence and a KpnI site in both the cloning and EST vectors, it was possible to reconstruct the full length honeybee FucTA sequence encoding a protein of 452 amino acids (M r 53 kDa), with 52% identity over 439 residues to Drosophila FucTA with a shorter putative stem region than the fly protein. The significance of the homology at the DNA level was also

  fucosyltransferase expression in order to perform screening for the ability of an open reading frame to confer anti-HRP binding in vivo, i.e. to prove the specific modification of glycoproteins of cells transfected with core α1,3-fucosyltransferases[START_REF] Rendić | Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells[END_REF] 29]. In the present study, we chose Sf9 cells (a typical biotechnological lepidopteran cell line), which naturally display only a low degree of binding to anti-HRP. Induction of core α1,3-fucosylation upon transfection of relevant open reading frames in this cell line is expected to result in the creation of the anti-HRP epitope, which can then be detected by Western blotting. We, therefore, transfected these cells with the honeybee α1,3-fucosyltransferase homologues and employed Drosophila FucTA as a positive control; vectors containing the entire open reading frame encoding each enzyme, with no tag, were used. As shown in Figure7B, of the honeybee fucosyltransferases, only the FucTA conferred the ability to bind anti-HRP to glycoproteins of Sf9 cells. The lanes with the cells transfected with the honeybee FucTB and

Figure 1 :

 1 Figure 1: Comparison of N-linked oligosaccharides from mammals, insects and plants: A.Typical mammalian 'complex' N-glycan structure containing terminal sialic acids and α1,6fucose linked to the proximal N-acetylglucosamine. B. N-glycan structures characteristic for insects. The MM and MMF6 structures are abundant, whereas the immunogenic MMF 3 F 6 structure accounts for 1% of the N-glycans from Drosophila. C. Typical structure found in plants. The plant N-glycans share some features with insect N-glycans, especially the α1,3linked fucose. D. Most complex N-glycan structure found on bee venom glycoproteins; apart from the immunogenic α1,3-fucose linked to the proximal N-acetylglucosamine, this structure contains a second α1,3-fucose linked to the N-acetylglucosamine creating a Lewis-like structure. As shown in this study, the synthesis of these structures is catalysed by FucTA and FucTC, respectively. E. Blood group related Lewis X structure present in mammals. "-R" represents an oligosaccharide backbone of a glycoconjugate. The glycans are depicted following the glycan nomenclature of the Consortium for Functional Glycomics (http://www.functionalglycomics.org) with the mammalian structure also displayed in an extended IUPAC/IUBMB-type form.

Figure 2 :

 2 Figure 2: Synthesis and subsequent modifications of glycan structures used as substrates in enzymatic assays:The various substrates used in this study, except the dabsylated MMglycopeptide, were acceptors for either FucTA or FucTC. Products of these enzymes are shaded; glycans are depicted following the glycan nomenclature of the Consortium for Functional Glycomics (http://www.functionalglycomics.org). A. Dabsylated glycopeptides (MM, GnGn and βGNβGN) were synthesised from the dabsylated GalGal-glycopeptide using relevant enzymes. β1,4and β1,3-galactosidases were used to remove galactose residues, whereas jack bean hexosaminidase was used to remove the GlcNAc residues. Bovine β1,4-galactosyltransferase I, an enzyme known to also utilise UDP-GalNAc, was used to the synthesise the dabsylated βGNβGN-glycopeptide. B. Dansyl-GnGnF 6 , produced from an asialoagalacto-IgG glycopeptide, has been previously shown to be a substrate for core α1,3-fucosyltransferases from plants and insects. C. Lacto-N-neo-tetraose is a Lewistype fucosyltransferase substrate.

Figure 3 :

 3 Figure 3: Homologies of honeybee α1,3-fucosyltransferases: A. Alignment of the conserved regions of honeybee α1,3-fucosyltransferase homologues. Residues identical in all sequences are highlighted. The amino acid numbering starts from the putative first methionine and potential N-glycosylation sites are underlined. The motifs I and II defined by Oriol et al. [39] are also shown. The 'core' motif, containing the sequence SNCXXRN, appears to be present in those core α1,3-fucosyltransferases which require the prior action of N-acetylglucosaminyltransferase I (i.e., all plant and insect core α1,3-fucosyltransferases described to date, but not Caenorhabditis elegans FUT-1 which does not accept substrates with an α1,3-antennal terminal GlcNAc residue). B. Phylogenetic tree of insect and mammalian α1,3-fucosyltransferase homologues. The following protein sequences were analysed using ClustalW and NJPlot programs (http://www.ebi.ac.uk/clustalw/): AmA-C: Apis mellifera fucosyltransferases A, B and C; DmA-D: Drosophila melanogaster fucosyltransferases A, B, C and D; HsIII-XI: Homo sampiens fucosyltransferases III, IV, V, VI, VII, IX, X and XI; MmX-XI: Mus musculus fucosyltransferases X and XI; BmA-C5: Bombyx mori manually assembled fucosyltransferase homologues A (AADK01016505), B1 (AADK01001074), B2 (AADK01006340), C1 (AADK01004147), C2 (AADK01000551), C3 (AADK01017942), C4 (AADK01001565) and C5 (BAAB01004223); AeA/C: Aedes aegypti manually assembled fucosyltransferase homologues A (AAGE02021501) and C (AAGE02000340, AAGE02000341); AgA/C: Anopheles gambiae manually assembled fucosyltransferase homologues A (AAAB01008879) and C (AAAB01008846); GmC: Glossina morsitans fucosyltransferase homologue C (AJ582622); TcA-C2: Tribolium castaneum manually assembled fucosyltransferase homologue A (AAJJ01000602), B1 (AAJJ01000605), B2 (AAJJ01000026), C1 (AAJJ01000008) and C2 (AAJJ01000008).GenBank accession numbers of genomic DNA sequences which were used to manually assemble sequences are in brackets. Proteins, for which activity was demonstrated in vitro and/or in vivo, are appropriately labelled as core or Lewis-like enzymes.

Figure 4 :

 4 Figure 4: Activity of honeybee fucosyltransferases expressed in Pichia analysed by MALDI-TOF-MS. Culture supernatants of yeast transformed with either FucTA (A), FucTB (B) or FucTC (C) were assayed using four dabsylated glycopeptides (MM, GnGn, GalGal and βGNβGN) in the presence of GDP-Fuc. The glycans are depicted following the glycan nomenclature of the Consortium for Functional Glycomics (http://www.functionalglycomics.org), i.e., mannose residues are shown as grey circles,

Figure 5 :

 5 Figure 5: Activity of honeybee fucosyltransferases expressed in Pichia analyzed by RP-HPLC. Culture supernatants of yeast transformed with either FucTA or FucTC were assayed using either dansylated-GnGnF 6 glycopeptide (FucTA) or lacto-N-neo-tetraose (FucTC) in

Figure 6 :

 6 Figure 6: Properties of recombinant honeybee FucTA and FucTC. Cation dependency, pH and temperature optima were determined using either ten-fold concentrated (FucTA) or unconcentrated (FucTC) honeybee enzymes expressed in Pichia. Relative activity was normalised based on the condition giving the highest rate of fucose transfer.

Figure 7 :

 7 Figure 7: Epitope formation by honeybee FucTA and FucTC: A. Honeybee FucTA and FucTC expressed in Pichia confer anti-HRP and anti-Le x staining to glycoproteins.Recombinant FucTA and FucTC were incubated with modified transferrin carrying Nglycans predominantly terminating with β1,2-GlcNAc (i.e., GnGn-), β1,4-Gal (i.e., GalGal-) or β1,4-GalNAc (i.e., βGNβGN-transferrin) residues. FucTA confers the anti-HRP staining to the used transferrin glycoforms (first row). FucTC but not FucTA confers anti-Le x (anti-L5) staining only to GalGal-transferrin (second row). B. Expression of honeybee fucosyltransferases in Sf9 insect cells. Cells transfected with either an empty pIZTV5/His vector (1), honeybee FucTA (2), honeybee FucTB (3), honeybee FucTC (4) and Drosophila FucTA (5) were lysed and subject to SDS-PAGE and Western blotting with anti-horseradish peroxidase.

Figure 8 :

 8 Figure 8: Expression patterns of fucosyltransferases in different tissues and glands of A.mellifera. Total RNA was isolated from the whole larva (L) as well as from thorax muscles (M), brains (B), venom glands (Vg) and hypopharyngeal glands (Hg) of nurse honeybees or of foragers from the same colony. RT-PCR was performed with the primers specific for individual FucTs (see Experimental procedures) using different numbers of cycles. In the

Figure 1

 1 Figure 1

Biochemical Journal Immediate Publication. Published on 10 Oct 2006 as manuscript BJ20060964

Acknowledgements

The authors thank Dr. Hubert Paschinger for the gift of male honeybee larvae, Dr. Melitta Schachner and Dr. Gabriele Loers (Universität Hamburg) for the L5 anti-Le x monoclonal antibody, Dr. Jozef Juršík for honeybees, Dr. Gene Robinson (University of Illinois, Urbana, IL, USA) for the bee brain EST clones and Thomas Iskratsch for preparing the transferrin neoglycoforms. This work was supported by grants from the Fonds zur Förderung der wissenschaftlichen Forschung (P17681) to IBHW and by a grant from the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences VEGA 2/5074/25 to Dr. Ján Mucha.

The abbreviations used are: AMPD, 2-amino-2-methyl-1,3-propanediol; anti-HRP, antiserum


Adriana Badarau

Christian Damblon

Michael I Page
email: m.i.page@hud.ac.uk

The Activity of the Dinuclear Cobalt-β-Lactamase from Bacillus cereus in Catalysing the Hydrolysis of β-Lactams

Keywords: Metallo-β-lactamases, Cobalt Substitution, pH-rate magnetic resonance, EPR, electron paramagnetic resonance, PAC, perturbed angular correlation, LMCT, ligand to metal charge transfer, MCD, magnetic circular dichroism

Metallo-β-lactamases are native zinc enzymes that catalyse the hydrolysis of β-lactam antibiotics, but are also able to function with cobalt (II) and require one or two metal-ions for catalytic activity. The hydrolysis of cefoxitin, cephaloridine and benzylpenicillin catalysed by cobalt substituted β-lactamase from Bacillus cereus (BcII) has been studied at different pHs and metalion concentrations. An enzyme group of pK a 6.52±0.1 is found to be required in its deprotonated form for metal-ion binding and catalysis. The species that results from the loss of one cobalt ion from the enzyme has no significant catalytic activity and is thought to be the mono-nuclear CoBcII. It appears that di-nuclear CoBcII is the active form of the enzyme necessary for turnover, while the mono-nuclear CoBcII is only involved in substrate binding. The cobalt enzyme is a more efficient catalyst for the hydrolysis of some β-lactam antibiotics suggesting that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarise the carbonyl group and stabilise the oxyanion tetrahedral intermediate.

Introduction

The major bacterial defence mechanism against the normally lethal action of β-lactam antibiotics consists of bacteria producing a class of enzymes, the β-lactamases, which catalyse the hydrolysis of the β-lactam ring, rendering the drugs inactive (Scheme 1). From the mechanistic point of view, β-lactamases are divided into two classes: serine β-lactamases, (classes A, C and D β-lactamases), which use an active site serine residue for hydrolysis, and metallo-β-lactamases, (MBLs), (class B β-lactamases), which require one or two zinc ions for catalytic activity [START_REF] Frère | Beta-lactamases and bacterial resistance to antibiotics[END_REF].

MBLs have been further divided into three subclasses, B1, B2 and B3, based on their amino acid sequences, substrate profiles and metal-ion requirement [START_REF] Galleni | Standard numbering scheme for class B β-lactamases[END_REF]. Subclass B1 of MBLs is the largest and contains four well-studied β-lactamases: BcII from Bacillus cereus [START_REF] Fabiane | Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Orellano | Spectroscopic characterization of a binuclear metal site in Bacillus cereus β-lactamase II[END_REF][START_REF] Paul-Soto | Mono-and binuclear Zn 2+ -β-lactamase. Role of the conserved cysteine in the catalytic mechanism[END_REF], CcrA from Bacteroides fragilis [START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis[END_REF][START_REF] Paul-Soto | Mono-and binuclear Zn 2+ -β-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions[END_REF][START_REF] Yang | Kinetic properties and metal content of the metallo-β -lactamase CcrA harboring selective amino acid substitutions[END_REF][START_REF] Wang | On the mechanism of the Bacteroides fragilis metallo-β-lactamase[END_REF], IMP-1 from Pseudomonas aeruginosa [START_REF] Laraki | Biochemical characterisation of the Pseudomonas aeruginosa 101/1477 metallo-β-lactamase IMP-1 produced by Escherichia coli[END_REF][START_REF] Haruta | Functional analysis of the active site of a metallo-β-lactamase proliferating in Japan[END_REF][START_REF] Concha | Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor[END_REF] and BlaB from Chryseobacterium meningosepticum [START_REF] Garcia-Saez | The 1.5-A structure of Chryseobacterium meningosepticum zinc β-lactamase in complex with the inhibitor, D-Captopril[END_REF]. They efficiently hydrolyze a wide range of substrates, including penicillins, cephalosporins and carbapenems [START_REF] Crowder | Structure and function of metallo-β-lactamases[END_REF]. The most common representatives of subclass B2 are CphA from Aeromonas hydrophila [START_REF] Hernandez Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-β-lactamase activity and stability[END_REF] and ImiS from Aeromonas veronii [START_REF] Crawford | Spectroscopic studies on cobalt(II) -substituted metallo-β-lactamase ImiS from Aeromonas veronii bv[END_REF], which preferentially hydrolyze carbapenems, e.g., imipenem and meropenem [START_REF] Rasmussen | Carbapenem hydrolysing β-lactamases[END_REF], and have poor activity against penicillins and cephalosporins [START_REF] Felici | An overview of the kinetic parameters of class B β-lactamases[END_REF][START_REF] Felici | Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-βlactamases[END_REF]. Finally, subclass B3 contains the only known tetrameric zinc β-lactamase, the L1 enzyme from Stenotrophomonas maltophilia [START_REF] Crowder | Overexpression, purification, and characterization of the cloned metallo-β-lactamase (L1) from Stenotrophomonas maltophilia[END_REF], and the monomeric FEZ-1 from Legionella gormanii [START_REF] Mercuri | Biochemical characterization of the FEZ-1 metallo-β-lactamase of Legionella gormanii ATCC 33297 T produced in Escherichia coli[END_REF]. Both enzymes hydrolyze a wide spectrum of β-lactam antibiotics [START_REF] Felici | An overview of the kinetic parameters of class B β-lactamases[END_REF][START_REF] Felici | Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-βlactamases[END_REF].

There are two zinc binding sites [START_REF] Galleni | Standard numbering scheme for class B β-lactamases[END_REF][START_REF] Fabiane | Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Orellano | Spectroscopic characterization of a binuclear metal site in Bacillus cereus β-lactamase II[END_REF][START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis[END_REF][START_REF] Concha | Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor[END_REF][START_REF] Garcia-Saez | The 1.5-A structure of Chryseobacterium meningosepticum zinc β-lactamase in complex with the inhibitor, D-Captopril[END_REF] but their occupancy has different effects in different MBLs. Furthermore, the zinc ligands in the two sites are not the same and are not fully conserved between the different MBLs. In the B1 enzymes, such as the Bacillus cereus MBL, BcII, (Figure 1) the zinc in site 1 (the histidine site or His 3 site) is four-coordinated through the nitrogens of the imidazoles of three histidine residues (116, 118, and 196) and a water molecule, Wat 1 . The zinc in the second site (site 2, the DCH or the Cys site) is coordinated to five ligands -His263, Asp120, Cys221 and one water molecule, carbonate [START_REF] Carfi | 1.85 A resolution structure of the zinc (II) β-lactamase from Bacillus cereus[END_REF] or another water, often referred to as the apical water, or Wat 2 [START_REF] Fabiane | Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis[END_REF][START_REF] Carfi | X-ray structure of the Zn(II) β-lactamase from Bacteroides fragilis in an orthorhombic crystal form[END_REF][START_REF] Concha | Crystal structure of the cadmium-and mercury-substituted metallo-β-lactamase from Bacteroides fragilis[END_REF]. The two metal-ions are relatively close to each other, but the distance between them varies from 3.4 to 4.4 Å in different structures of the BcII and CcrA enzymes [START_REF] Fabiane | Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis[END_REF][START_REF] Carfi | 1.85 A resolution structure of the zinc (II) β-lactamase from Bacillus cereus[END_REF][START_REF] Carfi | X-ray structure of the Zn(II) β-lactamase from Bacteroides fragilis in an orthorhombic crystal form[END_REF][START_REF] Concha | Crystal structure of the cadmium-and mercury-substituted metallo-β-lactamase from Bacteroides fragilis[END_REF]. The CcrA enzyme shows a bridging water ligand between the two metals that is thought to exist as a hydroxide ion [START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis[END_REF][START_REF] Concha | Crystal structure of the cadmium-and mercury-substituted metallo-β-lactamase from Bacteroides fragilis[END_REF] as does BcII at pH 7.5 [START_REF] Paul-Soto | Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus β-lactamase II: a perturbed angular correlation of γ-rays (PAC) spectroscopy study[END_REF] although at lower pH this solvent molecule is strongly associated to the zinc in site 1 [START_REF] Fabiane | Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Carfi | The 3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein fold[END_REF].

MBLs have different metal-ion affinities for the two binding sites and apparently different metal-ion requirement for catalysis. The class B1 enzyme BcII, from Bacillus cereus, has very different dissociation constants for the two metal binding sites, based on fluorescence spectroscopy using a chromophoric chelator. For the loss of metal-ion from the mono-nuclear enzyme, the dissociation constant, K mono , is 6.2x10 -10 M and that for the loss of one metal-ion from the di-nuclear MBL, K di , is 1.5x10 -6 M [START_REF] De Seny | Metal ion binding and coordination geometry for wild type and mutants of metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII); a combined thermodynamic, kinetic and spectroscopic approach[END_REF]. K mono decreases significantly, from nM to pM, in the presence of substrate whereas K di decreases by only two fold [START_REF] Wommer | Substrate activated zinc binding of metallo-β-lactamases; physiological importance of the mononuclear enzymes[END_REF]. This suggested that the mono-zinc enzyme is responsible for the catalytic activity under physiological conditions, where the concentration of free Zn 2+ is in the pM region [START_REF] Wommer | Substrate activated zinc binding of metallo-β-lactamases; physiological importance of the mononuclear enzymes[END_REF]. Conversely, and despite the very close similarity with BcII, the class B1 enzyme CcrA, from Bacteroides fragilis, binds both zinc ions very tightly [START_REF] Crowder | Characterization of the metal-binding sites of the β-lactamase from Bacteroides fragilis[END_REF]. Kinetic studies of CcrA with nitrocefin have shown that only the di-nuclear species is active and that a previously observed 'mono-zinc' CcrA enzyme [START_REF] Paul-Soto | Mono-and binuclear Zn 2+ -β-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions[END_REF] was a mixture of the di-zinc and the apo (metal free) enzyme [START_REF] Fast | Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-β-lactamase from Bacteroides fragilis[END_REF].

Based on the pH-dependence of k cat /K m for the ZnBcII catalysed hydrolysis of benzylpenicillin and cephaloridine, and of the competitive inhibition constant (K i ) of BcII with a thiol inhibitor, it has been proposed that there are three catalytically important groups in the enzyme: two of pK a 5.6 which were assigned to Asp-120 and the zinc bound water, and are required in their deprotonated form for activity, and one of pK a 9.5 which is required in its protonated form for maximum activity [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF]. Increasing the metal-ion concentration (from 10 -6 to 10 -3 M) had no effect on the rate constant at neutral pH but restored the enzyme activity at lower pHs (pH≤5.5), with a corresponding decrease of the apparent pK a values (from 5.6 to 4.5). Decreasing the pH liberates metal-ion from the enzyme due to protonation of one of the metal ligands. In terms of the equilibrium between mono-and di-nuclear enzymes, binding of the second zinc ion lowers the pK a s of the catalytically important groups on the acidic limb [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF]. The proposed mechanism involves the metal bound hydroxide attacking the carbonyl to generate a mono-anionic tetrahedral intermediate, which is followed by proton abstraction by the Asp120 to give a dianionic species (Scheme 2). It was suggested that the same aspartic acid residue then functions as proton donor, to facilitate C-N bond fission and opening of the β-lactam ring [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF].

Changing the metal-ion in zinc enzymes gives the opportunity to explore the structural and mechanistic role of the metal [START_REF] Auld | Removal and replacement of metal ions in metallopepatidases[END_REF][START_REF] Vila | Alkaline transition of Rhus vernicifera stellacyanin, an unusual Blue copper protein[END_REF][START_REF] Bertini | Strategies of signal assignments in paramagnetic metalloproteins. An NMR investigation of the thiocyanate adduct of the cobalt (II) substituted human carbonic anhydrase II[END_REF][START_REF] Oz | NMR spectroscopic studies of I=1/2 metal ions in biological systems[END_REF][START_REF] Bennet | EPR studies on the mono-and dicobalt(II) -substituted forms of the aminopeptidase from Aeromonas proteolytica. Insight into the catalytic mechanism of dinuclear hydrolases[END_REF][START_REF] Bauer | Coordination geometry for cadmium in the catalytic zinc site of horse liver alcohol dehydrogenase: studies by PAC spectroscopy[END_REF]. The zinc of metallo β-lactamases, can be substituted with cadmium, cobalt and manganese to give catalytically active enzymes [START_REF] Paul-Soto | Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus β-lactamase II: a perturbed angular correlation of γ-rays (PAC) spectroscopy study[END_REF][START_REF] Bicknell | The pH-dependence of class B and class C β-lactamases[END_REF][START_REF] Baldwin | Production of a variant of beta-lactamase II with selectively decreased cephalosporinase activity by a mutant of Bacillus cereus 569/H/9[END_REF]. The metal environment of cobalt substituted metallo β-lactamases has been studied by UV-Vis spectroscopy, NMR and EPR [START_REF] Orellano | Spectroscopic characterization of a binuclear metal site in Bacillus cereus β-lactamase II[END_REF][START_REF] Crowder | Characterization of the metal-binding sites of the β-lactamase from Bacteroides fragilis[END_REF][START_REF] Wang | Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-β-lactamase that provides multiple antibiotic resistance[END_REF][START_REF] Myers | Production, purification and spectral properties of metal-dependent β-lactamase from Bacillus cereus[END_REF][START_REF] Garrity | Direct evidence that the reaction intermediate of metallo-β-lactamase L1 is metal bound[END_REF][START_REF] Crawford | Over-expression, purification, and characterization of metallo-βlactamase ImiS from Aeromonas veronii bv[END_REF]. The kinetic effect of cobalt substitution in BcII is to make the progress curves for the hydrolysis of benzylpenicillin biphasic, with an initial burst followed by a transient to a steady-state rate [START_REF] Bicknell | Cryoenzymology of Bacillus cereus β-lactamase II[END_REF][START_REF] Bicknell | Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus β-lactamase II[END_REF]. The size of the burst was greater than the concentration of enzyme, which led the authors to propose a branched kinetic hydrolysis pathway involving two enzyme-substrate intermediates, ES 1 and ES 2 , which we have recently reinterpreted as mono-and di-nuclear forms of the enzyme [46]. The pH-dependencies of the Michaelis-Menten constants k cat /K m and k cat for the Co-BcII catalysed hydrolysis of benzylpenicillin were performed at saturating concentrations of cobalt ion [START_REF] Bicknell | The pH-dependence of class B and class C β-lactamases[END_REF]. However, given more recent studies [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF], which have shown that the pK a value of the ionisation on the acidic limb of the ZnBcII pH-rate profile is metal-ion concentration dependent, we have studied the pHrate profiles for CoBcII at different cobalt ion concentrations. Cobalt has about 100 fold less affinity than zinc for the two binding sites of BcII, i.e., the dissociation constants are higher, which makes it easier from an experimental point of view, for studying the differences between the catalytic properties of the mono-and the di-nuclear forms of the enzyme.

Experimental

Materials

Reagents used in all kinetic experiments were analytical or an equivalent grade. Cephaloridine was supplied by GlaxoSmithKline and used without further purification. Buffers, benzylpenicillin, cefoxitin, Chelex 100 and CoCl 2 (99.9999%) were purchased from Sigma.

Deionised ultra pure water (18 MΩcm) was used for the preparation of buffers and other aqueous solutions. The buffers used were: acetate (pK a 4.75), MES (pK a 6.15), MOPS (pK a 7.20), TAPS (pK a 8.40), CHES (pK a 9.2). Buffer solutions were prepared just prior to the experiment, and their ionic strength was kept constant by means of potassium chloride.

The apo Bacillus cereus 569/H/9 enzyme (metal free BcII) was prepared by the following procedure: ZnBcII (prepared and purified as described [47]) was dialyzed, whilst stirring, against two changes of 0.015M MES, pH=6.5, containing 0.1M NaCl and 0.02M EDTA over a 12h period; EDTA was removed from the resulting apoenzyme solution by four dialysis steps against the same buffer containing 1M NaCl and Chelex 100 and finally two dialysis steps against 0.015M MES, pH=6.5 containing 0.1M NaCl and Chelex 100. The resulting apoenzyme contained less than 3% Zn 2+ , as determined by atomic absorption spectroscopy, and less than 10% free EDTA, as shown by 1 H-NMR. Copyright 2006 Biochemical Society Equipment pH Measurements were made using a ф40 pH meter (Beckman, Fullerton, U.S.A.) with a calomel glass electrode (Beckman). A two point calibration of the pH meter was taken at 30 0 C prior to use, with a pH 7 phosphate "green" buffer (Beckman) and a pH 4 or pH 10 calibration buffer (BDH, Poole, U.K.). UV spectrometry was carried out on a Cary 1E UV-Visible spectrometer equipped with a twelve compartment cell block thermostated by using a peltier system (Varian, Australia). Rate constants were estimated using the Cary Win UV kinetics application Version 02.00 [START_REF] Carfi | The 3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein fold[END_REF].

The concentration of free EDTA in the apo-enzyme solution was determined by 1 H-NMR spectra on a 400MHz instrument (Brucker, Germany), using the standard addition method.

The residual zinc content of apoBcII was determined by atomic absorption spectroscopy on a Perkin Elmer AAnalyst 100 atomic absorption spectrometer. The hollow cathode lamp wavelength was set at 213.9nm, with a current of 7mA, and a slit wave of 0.7nm. Zinc sulphate solutions of different concentrations were used as standards.

General kinetic procedure

In a typical experiment, the apoBcII (1x10 -7 -2x10 -6 M) was incubated for 5 min at 30 0 C in the buffer (2ml) containing the corresponding cobalt ion concentration (10 -4 -10 -3 M), in a quartz cuvette (200-2500nm, Hellma). Unless otherwise specified, the reaction was initiated by adding the substrate and was followed by the decrease in absorbance at 235nm for benzylpenicillin (Δε=820M -1 cm -1 ) and 260nm for cefoxitin (Δε=4000M -1 cm -1 ) and cephaloridine (Δε=8000M - 1 cm -1 ).

Steady-state kinetics

The Michaelis-Menten kinetic parameters, k cat , K m and k cat /K m were determined as follows.

Below saturation, where [S]<<K m , the curves were fitted to a simple first-order rate law to obtain the pseudo-first-order rate constants, k obs , which were shown to be first-order in enzyme concentration. The second-order rate constant k cat /K m was obtained by dividing k obs by the concentration of enzyme used. For the determination of k cat and K m , the substrate concentrations used were in the range of the K m values. The initial rates, measured for at least seven substrate concentrations, were fitted directly to the Michaelis Menten equation using SCIENTIST software (Micro Math Scientific Software, Utah, USA), to obtain the apparent k cat and K m values for each pH and each metal-ion concentration.

Results and Discussion

pH-rate profiles for Co-BcII catalysed hydrolysis of cefoxitin, cephaloridine and benzylpenicillin.

The kinetic studies of metallo-enzymes that involve the replacement of the native-enzyme should ensure that the contribution from any remaining wild-type enzyme to the measured activity is as small as possible. In this study, a background hydrolysis rate of all substrates (cefoxitin (1), cephaloridine (2) and benzylpenicillin (3)) in the presence of the apo-enzyme was always determined and was insignificant, unless otherwise stated. In the following experiments, two cobalt ion concentrations were used, 10 -3 M and 10 -4 M, which should ensure that the monocobalt species is always present and that a variable di-cobalt species could be present as the dissociation constants for loss of the metal-ion from the mono-(K mono ) and di-cobalt (K di ) species are 9.3x10 -8 M and 6.0x10 -5 M, respectively [START_REF] De Seny | Metal ion binding and coordination geometry for wild type and mutants of metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII); a combined thermodynamic, kinetic and spectroscopic approach[END_REF]. Under our experimental conditions at 30 0 C only clean first or zero order kinetics were observed over the entire progress curves for the hydrolysis of the substrates. The biphasic behaviour described in the introduction is only observed at low temperatures or low metal-ion concentrations [START_REF] Bicknell | Cryoenzymology of Bacillus cereus β-lactamase II[END_REF][START_REF] Bicknell | Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus β-lactamase II[END_REF][46]. For the three substrates studied here, cefoxitin (1), cephaloridine (2) and benzylpenicillin (3), the Michaelis constant, K m , is much lower for the cobalt substituted enzyme than for the native zinc enzyme. This makes it difficult to obtain kinetic data below saturation conditions in order to determine K m and k cat /K m , especially at lower pHs, as K m decreases with decreasing pH.

Cefoxitin (1)

The catalytic constant, k cat , for CoBcII catalysed hydrolysis of cefoxitin varies with pH and cobalt ion concentration as shown in Figure 2, indicative of activity being dependent on an ionisable group. Between pH 8 and 9 the reaction rate is pH-independent, but decreases at lower pHs. In the pH-rate dependent region, k cat decreases with a first-order dependence on hydronium ion concentration, i.e., the slope of log k cat against pH is unity. At low pH a 10 fold decrease in cobalt ion concentration decreases k cat by approximately 10 fold, whereas in the pH-rate independent region k cat changes less than three fold. The kinetically important ionisation in the k cat -pH profile has an apparent pK a value of 7 and 6.5 for CoBcII catalysed hydrolysis of cefoxitin at 10 -4 and 10 -3 M CoCl 2 , respectively (Figure 2).

Although the Michaelis constant, K m , could not be determined very accurately due to its very low value, especially at low pH, to a first approximation K m is also pH-independent above pH 8 and there is an evident trend of K m decreasing with decreasing the pH (Figure 3). The apparent sigmoidal dependence of pK m on pH generates apparent pK a values of 7.2 and 6.5, at 10 -4 M and 10 -3 M CoCl 2 , respectively. The values of K m may also be dependent on the concentration of metal-ion but the data is not definitive. At pH 8-9 and 5-6 there is little or no dependence on cobalt ion concentration but, between pH 6 and 7, K m does vary about 6-7 fold when the concentration of CoCl 2 varies from 10 -4 M to 10 -3 M (Figure 3).

Similarly, the values of the second-order rate constant, k cat /K m , for the CoBcII catalysed hydrolysis of cefoxitin, are subject to considerable error (due to the errors involved in determining the K m ) but show an apparent bell shape pH-rate profile, with a decrease in rate at high and low pH and an intermediate pH-independent region. The rate constant shows a firstorder dependence on both metal-ion and acid concentration at low pH, whereas in the pHindependent region, k cat /K m increases by less than 2 fold for a 10 fold increase in metal-ion concentration. The apparent pK a values for the acidic ionisation in the pH-k cat /K m profile are 6.3 and 5.8 for 10 -4 and 10 -3 M CoCl 2 , respectively.

Cephaloridine (2)

Using cephaloridine as a substrate, a similar trend for the variation of k cat , (Figure 4), K m and k cat /K m (data not shown) with pH at different CoCl 2 concentrations was observed. At low pH, the log k cat versus pH profile shows a first-order decrease of k cat with increasing hydronium ion concentration, which is again metal-ion concentration dependent, with apparent pK a values of 6.8 and 6.2 for 10 -4 and 10 -3 M CoCl 2 , respectively (Figure 4). The rate increases about 6 fold at low pH and less than 2 fold in the pH-independent region with increasing the CoCl 2 concentration from 10 -4 to 10 -3 M.

The decrease of K m with decreasing pH also appears metal-ion concentration dependent, and K m varies from about 3x10 -5 and 5x10 -5 M at high pHs to about 3x10 -6 and 8x10 -6 M at low pHs, at 10 -4 and 10 -3 M CoCl 2 , respectively. Whilst the quality of the data is poor, (due to the difficulties of attaining below saturation conditions), it is possible to see that the second-order rate constant, k cat /K m , decreases at pHs less than 6 and is metal-ion concentration independent in the pHindependent region. Unlike cefoxitin, there is no obvious evidence of a decrease in k cat /K m at higher pH for cephaloridine.

Benzylpenicillin (3)

For CoBcII catalysed hydrolysis of benzylpenicillin only the k cat versus pH profile has been determined at different metal-ion concentrations ([Co 2+ ] = 10 -3 M and 10 -4 M), (Figure 5), due to the very low K m values (<10 -4 M). At lower pHs, k cat decreases with decreasing metal-ion concentration, with apparent pK a values of 6.8 and 6.2 for 10 -4 M and 10 -3 M CoCl 2 , respectively.

As with the other substrates, at low pH, k cat shows a first-order dependence on acid and on metalion concentration, while in the pH-independent region, k cat increases by less than 2 fold on increasing the CoCl 2 concentration from 10 -4 and 10 -3 M (Figure 5).

Mechanistic models

The variation of k cat and k cat /K m with pH and cobalt ion concentration is similar for the three substrates used, which indicates that the phenomena are an intrinsic property of the enzyme and independent of the nature of the substrate. The decrease in the apparent pK a values with increasing cobalt ion concentration suggests that the inverse first-order effect of the acid concentration on the rate of hydrolysis at lower pHs is due to the protonation of a metal ligand, which is responsible for the loss of one cobalt ion from the enzyme active site, rather than to the protonation of a catalytically important group or to a change in the rate limiting step. The decrease in the values of k cat and K m with decreasing pH and metal-ion concentration suggests that there are at least two enzyme species, one of which has greater values of k cat and K m , and is dominant at high pH and metal-ion concentration. The other species results from the loss of a cobalt ion and has a lower K m and k cat , and is probably, effectively, inactive. Several possible explanations for the metal-ion concentration and pH-dependencies have been considered (Scheme 3).

In the model (i), the apo-enzyme is the dominant species present at low pH and low metal-ion concentration and is catalytically inactive, while the mono-cobalt enzyme is responsible for the catalytic activity. Even though this model explains the variation of k cat and k cat /K m with pH and metal-ion concentration, it does not explain the decrease in K m with decreasing pH (tighter substrate binding) as it has been shown that the apoenzyme does not bind the substrate, at least in the case of benzylpenicillin and some cephalosporins [48]. It follows that the enzyme species involved in the tight substrate binding (low K m ), at low pH and metal-ion concentration, is probably the mono-cobalt BcII. The observed rate dependence on cobalt ion concentration can be explained if the catalytically active species is formed by the uptake of one cobalt ion i.e. is a dicobalt enzyme species (models (ii), (iii) or (iv)). The increase in rate with increasing pH can be explained by the ionisation of a group involved in the binding of the second cobalt ion: this can be either the metal bound water (model ii) or a protein residue, in which case the bridging water can be either deprotonated (model iii) or protonated (model iv). The interconversion between mono-and di-cobalt enzyme species is also supported by the biphasic kinetics observed at lower temperatures [46].

A more detailed pathway involves the mono-cobalt deprotonated (ECo) enzyme, which forms at higher pHs, in substrate binding and possibly catalysis (Scheme 4). The experimental data for the three substrates were fitted to the equations ( 1)-( 2), which describe the variation of k cat and K m with pH and metal-ion concentration, according to Scheme 4. The data for the pK m -pH and logk cat -pH dependencies were fitted to equations ( 1) and ( 2) using the literature value of K di for K d , 6.0x10 -5 M [START_REF] De Seny | Metal ion binding and coordination geometry for wild type and mutants of metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII); a combined thermodynamic, kinetic and spectroscopic approach[END_REF], in order to obtain the substrate dependent parameters, K m , K m1 and K m2 , k cat , k cat1 and k cat2 and the substrate independent parameter, K a . It was found that the k cat values for the mono-cobalt enzyme, in both the protonated and deprotonated forms, (k cat1 and k cat2 , respectively), were close to zero (<0.1s -1 ). The only catalytically active species is the di-cobalt enzyme. For benzylpenicillin, for which only the values of k cat were available, the data were fitted to equation (1), fixing the value for K m as 1.6x10 -4 M. The derived constants for the three substrates are summarized in Table 1. The pK a value for the kinetically important ionisation was found to be 6.52±0.1. This pK a may correspond either to the cobalt bound water (hydroxide ion), whose protonation results in dissociation of a cobalt ion from the enzyme active site, or to a metal binding enzyme residue, such as Cys221. The pK a of Cys 221 (or Cys221-His263 pair) in the apoBcII was found to be 7.85 [46], but may decrease in the mononuclear species. It is commonly suggested that the role of the metal-ion in metalloproteases is to act as a Lewis acid by co-ordination to the peptide carbonyl oxygen giving a more electron deficient carbonyl carbon which then facilitates nucleophilic attack and stabilises the negative charge developed on the carbonyl oxygen of the tetrahedral intermediate anion. Another role of the metal-ion is to lower the pK a of the co-ordinated water so that the concentration of metal-bound 'hydroxide ion' is increased relative to bulk solvent hydroxide ion at neutral pH. This metal-bound conjugate base is also a better nucleophile than water, but obviously not better than 'free' hydroxide ion.

K Co H K K H K K K K Co H K K k H K K k K k k ] [ ] [ 1 ] [ 1 1 ] [ ] [ ] [ 2 2 1 2 2 2 1 1 + + + + + + ⋅ ⋅ + ⋅ + ⋅ ⋅ + ⋅ + = (1) d a m a m m d a a obs m K Co H K K H K K K K Co H K H K K ] [ ] [ 1 ] [ 1 1 ] [ ] [ ] [ 1
The dissociation of an acid with a low pK a generates a weaker base which usually corresponds to a weaker nucleophile. Hence the order of basic strength and so nucleophilicity is: aqueous hydroxide-ion, metal-bound hydroxide-ion then water. The better is the Lewis acid (i.e. a more electron deficient metal-ion centre) in stabilising the negative charge developed on the tetrahedral intermediate from the ß-lactam carbonyl oxygen, the greater will also be the effect on the acidity of bound water i.e. the lower the pK a . However, a lower pK a of zinc-bound water gives rise to a more weakly nucleophilic hydroxide-ion even though it may become the dominant species even at lower pH. Conversely, a weaker Lewis acid would be less effective at reducing the pK a of the zinc bound water but, at pHs above the pK a , would produce a more nucleophilic hydroxide-ion.

Similarly a high pK a water implies a weak Lewis acid and so the zinc ion will be less efficient at stabilising the tetrahedral intermediate. The main factors controlling the charge density on metalions with the same formal positive charge are ionic radius and the number and type of its ligands. Substitution of the native zinc in the metallo-β-lactamases by other metal-ions enables an exploration of different Lewis acidities on catalytic activity.

A comparison of the pH-k cat /K m profiles for cobalt substituted BcII species with that of the native zinc enzyme [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF], for the hydrolysis of cefoxitin and cephaloridine, shows the cobalt enzyme to have a higher maximum activity at their pH optimum and to be more active than ZnBcII at pH 7.

The cobalt enzyme is remarkably about 200 and 10 fold more active than the native ZnBcII with cefoxitin and cephaloridine as a substrate, respectively (Table 1). Assuming that the pK a of the cobalt bound water is 6.5 and that of the zinc bound water is 5.6 in BcII [START_REF] Bounaga | The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase[END_REF], then Co 2+ has a higher effective positive charge, so it is a weaker Lewis acid than Zn 2+ , but it gives a more nucleophilic metal-bound hydroxide ion. At pH 7, 96% of ZnBcII and 76% of CoBcII will be in their active deprotonated forms. The fact that the cobalt substituted BcII is more active than the native ZnBcII (Table 1) suggests that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarise the carbonyl group and stabilise the oxyanion tetrahedral intermediate.

8 Biochemical

 8

  Scheme 2

1 )Figure 2 .

 12 Figure 2. Plot of log k cat against pH for CoBcII catalysed hydrolysis of cefoxitin in 0.025M buffer, I=0.25M, in presence of 10 -4 M Co 2+ (x) and 10 -3 M Co 2+ (■); the solid lines are the calculated values using equation (1) and the parameters inTable 1 (Scheme 4).

Figure 3 .

 3 Figure 3. Plot of pK m against pH for CoBcII catalysed hydrolysis of cefoxitin in 0.025M buffer, I=0.25M, in presence of 10 -4 M Co 2+ (x) and 10 -3 M Co 2+ (■); the solid lines are the calculated values using equation (2) and the parameters in Table1(Scheme 4).

Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002

Table 1 .

 1 Calculated values of the rate and dissociation constants from Scheme 4, for CoBcII catalysed hydrolysis of cefoxitin (1), cephaloridine (2) and benzylpenicillin (3) in buffer, 0.025M, I=0.25M, at 30 0 C, (K d =6.0x10 -5 M). The corresponding values, determined in similar conditions for the native ZnBcII, are given for comparison.

			k cat /K m	k cat	K m	pK a	K m1	K m2
			(10 6 M -1 s -1 )	(s -1 )	(10 -6 M)		(10 -6 M)	(10 -6 M)
	1	Co 2+	0.052±0.009	16±2.0	310±10	6.52±0.1	12±1.0	100±20
		Zn 2+ 0.00027±0.00005 0.57±0.1	2100±200
	2	Co 2+	1.7±0.3	67±10	40±10	6.52±0.1	3.0±0.3	25±5.0
		Zn 2+	0.15±0.02	160±20	1060±150	5.5±0.05 a
	3	Co 2+	2.4±0.4	390±20	160±20	6.52±0.1	12±2.0	500±100
		Zn 2+	0.87±0.1	950±120	1100±200	5.8±0.05 a

Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002 Copyright 2006 Biochemical Society

	(i)	EH + Co ++			ECo + H +
		inactive					active
	(ii)	E-Co-OH 2 +	Co ++		E-Co-OH-Co + H +
		inactive					active
	(iii)	EH-Co-OH + Co ++		E-Co-OH-Co + H +
		inactive					active
	(iv) EH-Co-OH 2 + Co ++		E-Co-OH 2 -Co + H +
		inactive					active
			Scheme 3
	EH 2 +Co	EHCo + S	K m1	EHCoS	k cat1	EHCo + P
			H +	K a
			ECo	+ S	K m2	E C o S	k cat2	ECo + P
			K d	Co
			ECo 2 + S	K m	ECo 2 S	k cat	ECo 2 + P
					Scheme 4
							21

Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002 Copyright 2006 Biochemical Society FIGURES His 169 His 118 His 116 HO H 2 O Asp 120 Cys 221 His 263

  Schematic representation of the two zinc binding sites in the metallo-β-lactamase from Bacillus cereus, BcII.

	-
	Zn 2+	Zn 2+
	Site 1	Site 2
	Figure 1.

Table 1 (

 1 Scheme 4).

22 Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002 Copyright 2006 Biochemical Society

Table 1 (

 1 Scheme 4). Plot of log k cat against pH for CoBcII catalysed hydrolysis of cephaloridine in 0.025M buffer, I=0.25M, in presence of 10 -4 M Co 2+ (x) and 10 -3 M Co 2+ (■); the solid lines are the calculated values using equation[START_REF] Frère | Beta-lactamases and bacterial resistance to antibiotics[END_REF] and the parameters in Table 1 (Scheme 4). Plot of log k cat against pH for CoBcII catalysed hydrolysis of benzylpenicillin in 0.025M buffer, I=0.25M, in presence of 10 -4 M Co 2+ (x) and 10 -3 M Co 2+ (■); the solid lines are the calculated values using equation (1) and the parameters in Table1(Scheme 4).

		2.2
		1.7
	log(k cat /s -1	0.7 1.2
		0.2
	-0.3
		5	6	7	pH	8	9

) Figure 4. ) Figure 5.

24 Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002

Biochemical Journal Immediate Publication. Published on 11 Sep 2006 as manuscript BJ20061002 Copyright 2006 Biochemical Society

This research was supported by the European Union research network on metallo-β-lactamases within the Training and Mobility of Researchers (TMR) Program, contract number HPRN-CT-2002-00264 and the University of Huddersfield