Glycobiology Advance Access originally published online on July 31, 2006
Glycobiology 2006 16(11):1021-1032; doi:10.1093/glycob/cwl029
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Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans
2 Equipe Glycobiologie Marine, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie-Paris6, Unité Mixte de Recherche 7139, Station Biologique, F-29682 Roscoff Cedex, Bretagne, France; 3 Service Commun de Résonance Magnétique Nucléaire, Université de Bretagne Occidentale, Brest, Bretagne, France; and 4 Laboratoires Goëmar, Saint Malo, Bretagne, France
1 To whom correspondence should be addressed; e-mail: gurvan{at}sb-roscoff.fr
Received on December 5, 2005; revised on June 23, 2006; accepted on July 18, 2006
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Abstract |
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Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an
-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared 
25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the -(1
4) glycosidic linkages within the blocks of repeating motifs [4)--L-fucopyranosyl-2,3-disulfate-(13)--L-fucopyranosyl-2-sulfate-(1]n. Key words: brown algae / Flavobacteriaceae / fucanase / NMR / sulfated polysaccharide
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Introduction |
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Marine macroalgae synthesize a great diversity of polysaccharides, which are used as cell wall constituents and for energy storage. Like land plants, these eukaryotic phyla produce neutral polysaccharides such as cellulose and starch. However, marine algae are characterized by their abundance of sulfated polysaccharides that have no equivalent in land plants (Kloareg and Quatrano, 1988
). The red algae produce sulfated galactans, agars, and carrageenans, which are commercially used for their gelling properties. The structures of agars and carrageenans have been extensively studied to understand and manipulate their physicochemical properties. The cell walls of brown algae also contain sulfated polysaccharides known as sulfated fucans. These anionic polymers encompass a continuous spectrum of highly ramified polysaccharides, ranging from high uronic acid, low-sulfate–containing polymers with significant proportions of D-xylose, D-galactose, and D-mannose to highly sulfated homofucan molecules with approximately three substituents per disaccharide (Kloareg and Quatrano, 1988; Mabeau et al., 1990). In the cell wall matrix, they associate with alginate, a linear polymer of ß-D-mannuronic acid and -L-guluronic acid (Kloareg and Quatrano, 1988). Sulfated fucans exhibit various biological activities (Berteau and Mulloy, 2003), both as cell wall ligands and as anticoagulant (Kropf et al., 1988; Goodner and Quatrano, 1993; Pereira et al., 1999), antiviral, and antiproliferative agents. They were also shown to be recognized as fortuitous, defense elicitors in tobacco, where they induce systemic resistance against tobacco mosaic virus (Klarzynski et al., 2003). These properties have attracted a renewed interest to their fine chemical structure in the last decade. Sulfated fucan oligosaccharides of 8–14 fucose units, with a regular repeating disaccharide structure built on two residue types, 2-sulfated fucose and 2,3-disulfated fucose, and with -(13) and -(14) glycosidic linkages, were isolated by chemical methods (Chevolot et al., 2001), indicating a significant degree of repetitiveness in the sulfated fucan backbone. Yet the general complex structure of sulfated fucans has remained somewhat elusive.
The algal polysaccharides constitute a crucial carbon source for many marine bacteria. Those microorganisms degrade the cell walls of marine algae by secreting specific glycoside hydrolases (Michel et al., 2006). We have so far isolated and structurally characterized several bacterial enzymes that depolymerize the sulfated galactans of red algae, the family GH16 ß-agarases and 
-carrageenases (Michel, Chantalat, Duee, et al., 2001; Allouch et al., 2003; Jam et al., 2005), and the 
-carrageenases, which constitute a distinct family of glycoside hydrolases, the family GH82 (Barbeyron et al., 2000; Michel, Chantalat, Fanchon, et al., 2001; Michel et al., 2003). Both the - and -carrageenases have been recently used in combination with liquid chromatography and mass spectrometry techniques to analyze the complex structure of carrageenans (Antonopoulos, Favetta, et al., 2005; Antonopoulos, Hardouin, et al., 2005). In this context, the isolation and expression of hydrolases degrading sulfated fucans would be of special interest to further elucidate the fine chemical structure of these polysaccharides and to shed light on the structure of hydrolases that degrade polysaccharides with high linear densities of sulfate-ester substituents. To date, only two highly similar sulfated fucan hydrolases, referred to as Fda1 and Fda2, were cloned and expressed from the marine bacterium Alteromonas sp. SN-1009. These homologous enzymes have been patented (Takayama et al., 2002), but they were not subjected to a detailed biochemical analysis.
On the basis of its capacity to degrade sulfated fucans from fucoid algae, we recently isolated a flavobacteriacean strain, SW5, which secretes sulfated fucan hydrolase activity in its culture medium (Descamps et al., 2006). A crude enzyme preparation from SW5 culture supernatant was used to degrade the sulfated fucans from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of [4)--L-fucopyranosyl-2,3-disulfate-(13)--L-fucopyranosyl-2-sulfate-(1]n, with the 3-linked residues at the nonreducing end (Descamps et al., 2006). We here report the purification, the cloning, and the overexpression of the sulfated fucan hydrolase from SW5, referred to as the fucanase FcnA. The protein consists of an N-terminal catalytic domain followed by three immunoglobulin-like repeats, and it harbors a C-terminal domain conspicuous of proteins from Bacteroidetes bacteria. The enzyme depolymerizes sulfated fucans according to an endolytic mode of action and cleaves the -(14) glycosidic linkage between 2-sulfated fucose and 2,3-disulfated fucose residues. We also show that, together with Fda1 and Fda2, FcnA belongs to a new family of glycoside hydrolases (Henrissat and Bairoch, 1996).
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Purification of an extracellular sulfated fucan-degrading enzyme from SW5
The purification of the sulfated fucan-degrading activity of the SW5 strain was based on a carbohydrate-polyacrylamide gel electrophoresis (C-PAGE) assay of the release of anionic oligosaccharides from the sulfated fucan fraction referred to as FS28 (Descamps et al., 2006
). From the concentrated bacterial culture supernatant, purification to electrophoretic homogeneity was achieved in four steps: protein precipitation with ammonium sulfate at 40–60% saturation, hydrophobic interaction chromatography on Phenyl Sepharose, ion-exchange chromatography on diethylaminoethyl (DEAE) Sepharose, and gel filtration on Superdex 200. The active protein fractions corresponded to 50, 35, 0.50, and 0.008% of the initial protein content, respectively. The latter protein fraction migrated as a single band in sodium dodecyl sulfate (SDS)–PAGE analysis, corresponding to an apparent molecular weight of 105 kDa (Figure 1). This sulfated fucan-degrading enzyme is referred to as the fucanase FcnA. The SDS–PAGE 105-kDa protein band was excised, and three internal peptide sequences were determined by Edman degradation, ITVDHVAGFTNLGNGAP, TSGPDWLTIQQTDANS, and TANTTYGINTVASM, referred to as A, B, and C, respectively.
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The fucanase gene fcnA
An internal gene probe of 203 nt was generated by polymerase chain reaction (PCR) from the peptidic microsequences A and B and used to screen a genomic library prepared with SW5 total DNA. Of the 6000 clones in the library, three clones, referred to as pAT153fu11, pAT153fu14, and pAT153fu33 and with respective insert sizes of 4.0, 13.5, and 4.0 kb, hybridized with the probe. Southern blot experiments showed that SW5 strain contains only one copy of the fcnA gene. Restriction analyses indicated that the inserts from pAT153fu11 and pAT153fu33 were the same genomic DNA fragment, cloned in opposite directions, and that the pAT153fu14 insert overlapped these fragments by 1.5 kb. The pAT153fu11 and pAT153fu14 inserts were sequenced on both strands. The two sequences perfectly overlapped over a length of 1610 nt, the fucanase open-reading frame (ORF) being interrupted at the 3' end of pAT153fu11 insert and finishing on the pAT153fu14 insert. The physical validity of this ORF was proven by PCR amplification from the genomic DNA, leading to the cloning of the entire, 3021 nt–long fucanase gene on a contig of 7485 nt. No obvious Shine-Dalgarno ribosome-binding site consensus sequence was found upstream of the fcnA gene (Kozak, 1999). In contrast, 15 nt upstream of the ATG codon, a conserved motif, AAxTAAAT, was present in the 5' end of fcnA. No obvious hairpin was identified at its 3' end as transcription terminator.
The fucanase FcnA displays a modular architecture
The predicted product of the fucanase gene from SW5 strain, FcnA, is a preprotein of 1007 amino acids, with a theoretical molecular mass of 110.3 kDa, consistent with the apparent molecular mass of the secreted protein on SDS–PAGE analysis, 105 kDa (Figure 1). It includes the three internal peptides determined by microsequencing. Based on the prediction program SignalP V2.0 (Nielsen et al., 1999), a signal peptide is likely to be cleaved between A28 and Q29. Using BLASTp and PSI-BLAST searches (Altschul et al., 1997), 60% of the sequence did not share any significant similarity with the protein sequences available in GENBANKnr. Only the core and the C-terminal end of FcnA brought significant matches. However, a BLASTp search of the PAT database (NCBI) provided hits with two patented fucanase sequences from Alteromonas sp. SN-1009 (Takayama et al., 2002), referred to as Fda1 (AAO00508
[GenBank]
) and Fda2 (AAO00509
[GenBank]
) (E-values: 2E-9 and 2E-7, respectively). The three proteins shared a large domain in their N-terminal ends (380 residues long). Fda1 and Fda2 are highly similar, displaying 70% identity over their entire length, 814 and 881 amino acids, respectively, whereas they displayed 24 and 23% sequence pairwise identities (38 and 37% sequence similarity), respectively, with FcnA over their common N-terminal domain. To further assess the significance of these sequence similarities with FcnA, we compared the hydrophobic cluster analysis (HCA) plots of the N-terminal domain of Fda2 and FcnA (Figure 2) (Lemesle-Varloot et al., 1990). HCA plots revealed a similar distribution of the hydrophobic clusters, confirming that these domains share similar secondary-structure elements. The HCA score based on the correspondences shown in Figure 2 was 74%, a value >60%, which is considered as a significant threshold in this comparison procedure (Lemesle-Varloot et al., 1990).
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In the protein core, RADAR searches detected three repeated domains of 105 residues each (repeats R1, G419-D527; R2, G528-N633; and R3, G634-N737; Figure 3A), which share 31% (R1/R2), 27% (R1/R3), and 48% (R2/R3) pairwise sequence identities (Heger and Holm, 2000). In this region, searches on the Pfam database identified three consecutive He-PIg domains (PF05345) (Bateman et al., 2004). These domains are predicted to adopt an immunoglobulin fold. A BLASTp search in GENBANKnr using the three repeated domains identified mainly bacterial hypothetical conserved proteins, with various annotations such as PKD domain protein (NP_953122
[GenBank]
, 4E-13), putative immunoglobulin precursor (ZP_00525205, 2E-9), or RTX toxin (ZP_00315133, 4E-12). The matching regions are similar repeated domains with pairwise sequence identities ranging from 20 to 36% (from 35 to 52% sequence similarities). These matching domains were assigned to the He-PIg, cadherin, or PKD Pfam families, which all belong to an immunoglobulin-like-fold superfamily, the E-set Pfam clan (Bateman et al., 2004). It is noteworthy that such domains are also found associated with catalytic domains belonging to the families GH5 and GH18 of the glycoside hydrolases (trEMBL code: Q48C6, Q693B8).
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In the C-terminal region, a stretch of 75 residues displayed significant matches, ranging from 24 to 40% sequence identity (46–62% sequence similarities), with the C-terminal domains of several proteins, mainly polysaccharidases and proteases, exclusively from bacteria of the Bacteroidetes phylum, including the genera Porphyromonas, Cytophaga, Zobellia, Flavobacterium, Microscilla, and Rhodothermus. In particular, the YPNP motif at the beginning of these domain sequences is highly conserved (Figure 3B).
Altogether, FcnA consists of a 1007-amino acid-long protein (110.3 kDa) with a modular architecture. This protein encompasses a 28-amino acid-long signal peptide, a 390-amino acid-long N-terminal domain, three successive He-PIg domains with 105 residues each, followed by a region of 196 amino acids without any significant sequence similarity with known proteins, and finally by a 75-amino acid-long C-terminal domain.
Overexpression of the fucanase FcnA gene from SW5 strain
The fucanase FcnA was expressed under three different forms. The first construct (FcnA1) encoded the whole FcnA sequence without its predicted signal peptide in frame with the N-terminal His-tag of the pDEST17 vector. The second construct (FcnA2) encompassed the N-terminal domain and the three He-PIg domains of the protein. The third construct (FcnA3) only contained the N-terminal domain. The expected products were proteins with 1001 residues (109.7 kDa), 788 residues (86.5 kDa), and 412 residues (46.6 kDa), respectively. SDS–PAGE analyses indicated that soluble products with the expected protein sizes were obtained from the FcnA1 and FcnA2 constructs, FcnA2 being expressed in a greater amount than FcnA1. Unfortunately, the shortest construct FcnA3 yielded an insoluble recombinant protein. Contaminant proteins co-eluted with FcnA1 and FcnA2 in the elution peak at 150 mM imidazole. Further purification attempts combining anion exchange and size-exclusion chromatography (SEC) did not significantly improve the purity of FcnA1 and FcnA2 (data not shown).
Action of fucanase FcnA on sulfated fucan fractions
The crude sulfated fucan fraction, FS28, was incubated in the presence of either one or the other partially purified recombinant enzymes, FcnA1 and FcnA2, and the enzymatic degradation kinetics was monitored by C-PAGE analysis and by spectrophotometry at 232 nm, the maximum wavelength for the absorbance of 4,5-unsaturated pyranoses. As illustrated for FcnA2 (Figure 4), both recombinant enzymes yielded the same profiles in C-PAGE analysis of the degradation products. The smallest oligosaccharide released by the fucanase approximately migrates at the level of a 
-neocarratetraose, which displayed three sulfate groups by disaccharide repeating unit (Guibet et al., 2006). No increase of absorbance at 232 nm was recorded as function of time, indicating that the degradation products are not unsaturated oligosaccharides. Therefore, FcnA is a glycoside hydrolase and not a polysaccharide lyase.
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However, as monitored by SEC, FcnA2 did not extensively degrade the FS28-sulfated fucan fraction, even after the removal of polyphenol contaminants by a chromatography step on Sephadex LH 20. Low-molecular-weight (LMW) components were also detected in the FS28 fraction by SEC analysis. To further purify the native polysaccharide, we ultrafiltrated the FS28 fraction onto a 50-kDa cutoff membrane (Millipore, Molsheim, France). As shown by SEC analyses on a Superdex 200 column connected to a Superdex peptide 10/300 GL column (GE Healthcare, Orsay, France), the retentate, referred to as FC, eluted at the exclusion volume and did not contain LMW contaminants (Figure 5B). 1H-NMR spectrum of the filtrate fraction showed signals typical of sulfated fucan substrate, especially in the regions of the -anomeric protons and of the methyl groups. But in comparison with the 1H-NMR spectrum of FC, these LMW components displayed additional, complex signals in the 3.1–4.9 ppm region, suggesting a substitution pattern different from that of the polymeric FC fraction (data not shown). In contrast to FS28, the size profile of FC was significantly modified on incubation with the recombinant fucanase FcnA2. The polymeric fraction significantly decreased, whereas two peaks eluted in the 74–94 mL region (Figure 5B). In the same elution conditions, purified -neocarratetraoses and -neocarrahexaoses, which displayed two sulfate groups by disaccharide repeating unit, eluted in the 84–94 mL region (Figure 5A, Guibet M., personal communication). The sulfated fucan fraction FCH+, resulting from the treatment of FC by mild acid hydrolysis, was amenable to a more complete degradation by the recombinant fucanase. In SEC analyses, the size profile of FCH+ was similar to that of FC, indicating that the mild acid hydrolysis did not result in the breakdown of the polymer (Figure 5C). After incubation in the presence of FcnA2, the size distribution was markedly shifted toward the lower molecular weights, with notably an increase of the first peak of oligosaccharides (Figure 5C).
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1H-NMR analyses of the enzymatic degradation of FCH+
As shown in Figure 6A, the 1H-NMR spectrum of sulfated fucan that had been subjected to mild acid hydrolysis, FCH+, was comparable with that of polymeric sulfated fucan. In the region of the anomeric protons (5.6–5.2 ppm), signals were found with chemical shifts similar to those described by Chevolot and others (2001) as assignable to alternating -(14)-linked 2,3-disulfated fucose and -(13)-linked 2-sulfated fucose residues (F2,3S-F2S). This pattern, which was proposed to be the main structure in the sulfated fucans from Ascophyllum nodosum and Fucus vesiculosus fucoid algae (Chevolot et al., 2001), is also present in the sulfated fucan of the fucalean alga P. canaliculata (Descamps et al., 2006). After incubating FCH+ with FcnA2, the reaction mixture was ultrafiltrated onto a 10-kDa cutoff membrane (Amicon) to separate the released oligosaccharides (filtrate) from the resistant fraction (retentate). In comparison with FCH+, the 1H-NMR spectrum of the filtrate was markedly modified (Figure 6B), particularly in the regions of the -anomeric protons (5.6–5.2 ppm) and of the methyl groups (1.4–1.0 ppm). In particular, the -anomeric signals corresponding to the repeating motif F2,3S-F2S were affected by the enzyme action. The signal assignable to the H1 of the internal -(13)-linked 2-sulfated fucose residues (5.30 ppm) was refined, but its intensity did not decrease on hydrolysis. In contrast, the signal of H1 of the internal -(14)-linked 2,3-disulfated fucose residues (5.45 ppm) was significantly diminished. A doublet also appeared, with a chemical shift close to that of the -(13)-linked 2-sulfated fucose reducing-end residues (5.51 ppm). Taken together, the 1H-NMR spectrum of the filtrate (Figure 6B) is very similar to that of the oligosaccharides 3 and 4 described by Descamp and others (2006), that is, -L-Fucp-2,3-diS-(13)--L-Fucp-2S-(14)--L-Fucp-2,3-diS-(13)--L-Fucp-2S-(14)--L-Fucp-2,3-diS-(1)3--L-Fucp-2S and -L-Fucp-2,3-diS-(13)--L-Fucp-2S-(14)--L-Fucp-2,3-diS-(13)--L-Fucp-2S.
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To further investigate the identity of the linkages cleaved by FcnA2, double quantum filtered-correlation spectroscopy (DQF-COSY) spectra were recorded on FCH+ and on the filtrate of FCH+ digested by FcnA2 (Figure 7). In the spectrum of FCH+ (Figure 7A), an H1–H2 cross-peak was identified for the internal -(14)-linked 2,3-disulfated fucose residues (cross-peak A, 5.45 and 4.69 ppm). This H1–H2 cross-peak was no longer present after the fucanase treatment, indicating the disappearance of internal -(14)-linked 2,3-disulfated fucose residues (Figure 7B). In contrast, the H1–H2 cross-peak of the internal -(13)-linked 2-sulfated fucose residues (cross-peak B, 5.30–5.35 and 4.62–4.65 ppm) was modified but remained apparent (cross-peak B'). Even though the signals for reducing- and nonreducing-end fucose residues were weak in the polymeric FCH+, an H1–H2 cross-peak was nevertheless identified for the reducing-end -(13)-linked 2-sulfated fucose residues (cross-peak C, 5.54 and 4.55 ppm). After the action of FcnA2, the intensity of this cross-peak clearly increased (cross-peak C'). The cross-peak D', which appeared at 5.4 and 4.6–4.65 ppm, was also assignable as the H1–H2 cross-peak of nonreducing-end -(14)-linked 2,3-disulfated fucose residues (Descamps et al., 2006). Altogether, the above results indicate that the filtrate fraction included oligosaccharides belonging to the series of the sulfated fucan oligosaccharides described by Descamp and others (2006). Therefore, FcnA cleaves the -(14) linkages within the repeating motifs [4)--L-fucopyranose-2,3-disulfate-(13)- -L-fucopyranose-2-sulfate-(1]n of the sulfated fucan polysaccharidic backbone.
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FcnA is a sulfated fucan
-1,4-endohydrolaseWe here report the purification of the fucanolytic activity secreted by a marine Flavobacteriaceae referred to as SW5 (Descamps et al., 2006
). Using a four-step procedure, the protein content was decreased 12,500-fold from the culture medium down to the enzyme electrophoretic homogeneity. Because the bioassay used to monitor protein purification, observation by C-PAGE of the release of oligosaccharides from a complex fucan substrate, was not amenable to quantification, the ratio of enzyme recovery was not assessed. Based on internal peptides sequenced from the purified protein, the enzyme was cloned and successfully produced in Escherichia coli, as both full-length and truncated proteins and tagged with an N-terminal polyhistidine tail. Several lines of evidence indicate that the full-length recombinant protein is indeed a fucanase: (1) its sequence featured the three internal peptide sequences found in the wild-type protein (I620-P636, T677-S692, and T743-M756); (2) based on C-PAGE analysis, it released oligosaccharides from the sulfated fucan fraction FS28 (Figure 4); (3) as seen by SEC (Figure 5) and NMR (Figures 6 and 7) analyses, the recombinant protein significantly depolymerized the purified sulfated fucan fractions FC and FCH+; and (4) the N-terminal domain of FcnA exhibited low yet significant sequence similarities with two patented fucanases (Takayama et al., 2002).
Based on the marked size decrease of the initial polysaccharide substrate (Figure 5) leading to the release of intermediary-sized fucan oligosaccharides (Figure 4), the fucanase proceeds according to an endo mode of action. However, FcnA did not extensively degrade the native sulfated fucan FS28, whereas it significantly degraded the purified fractions FC and FCH+. The difference of degradation susceptibility between FS28 and FC suggests that the LMW sulfated fucan components contained in FS28 may inhibit the fucanase. Moreover, the sulfated fucans of fucalean algae consist of a polysaccharidic backbone of alternating -(14)-linked fucose and -(13)-linked fucose residues, masked by various substitutions, mainly ester-sulfate substituents and lateral branches (Pereira et al., 1999; Chevolot et al., 2001; Bilan et al., 2004). The increased efficiency of FcnA on FCH+ indicates that the mild acid hydrolysis has likely removed some specific substituents, unmasking the sulfated fucan core structure and increasing the accessibility of fucanase to its substrate. For instance, mild acid hydrolysis has been reported to remove 2-sulfate esters at specific sites in the sulfated fucans from sea urchins (Pomin et al., 2005).
The 1H-NMR analyses of the oligosaccharides released by the enzymatic hydrolysis of FCH+ (Figures 6 and 7) indicate that they belong to the homologous series of the sulfated fucan oligosaccharides described by Descamp and others (2006). Therefore, FcnA cleaves (14) glycosidic linkages within the blocks of the repeating disaccharidic motif [4)--L-fucopyranosyl-2,3-disulfate-(13)--L-fucopyranosyl-2-sulfate-(1]n.
Sulfated fucan endohydrolases constitute a new family of glycoside hydrolases
FcnA is a modular enzyme featuring five domains, the N-terminal region, three consecutive He-PIg domains, and a C-terminal conserved domain. The C-terminal region of FcnA is conserved at the C-terminus of several proteins expressed by bacteria from the Bacteroidetes phylum, mainly including degrading enzymes such as polysaccharidases or proteases (Figure 3B). Nothing is known yet on the function of this specific peptidic sequence in these bacteria. Given the retention of fucan-degrading activity by the truncated form of the enzyme, FcnA2, this C-terminal domain does not participate in the catalytic machinery of FcnA. The three central repeats are predicted to adopt an immunoglobulin fold, but their exact functions remain elusive. Considering their small size and their association with other catalytic domains such as glycoside hydrolases of the families GH5 and GH18 (trEMBL code: Q48C6, Q693B8; Figure 3A), it is unlikely that these repeated domains possess a catalytic function.
The N-terminal region of FcnA, which was alignable over a length of 384 residues with the N-terminal domains of the 800 amino acid-long sulfated fucan hydrolases of Alteromonas sp. SN-1009, Fda1 and Fda2 (Figure 2), likely is the catalytic domain of the fucanase. This observation is further substantiated by the fact that the truncated protein FcnA2, which comprises the N-terminal domain and the three He-PIg repeats only, retained the enzyme activity. Our attempt to separately express the N-terminal domain resulted in an insoluble recombinant protein, suggesting an incorrect delineation of the domain limits. Another possibility is that the N-terminal domain interacts with the first He-PIg module and that it cannot be separated from this latter domain. Such an interaction has been observed between the catalytic domain and the carbohydrate binding module (CBM) module of the endo/exocellulase E4 (Sakon et al., 1997).
It follows that the N-terminal portions of Fda1 and Fda2, which share 24 and 23% sequence identity with that of FcnA, respectively, also are the catalytic domains of these fucanases. These three sequences do no match significantly with any proteins in the public databases, and no other fucanase sequences are known so far. We thus propose that they constitute a novel family of glycoside hydrolases (CAZy server: http://afmb.cnrs-mrs.fr/CAZY/) (Henrissat and Bairoch, 1996).
In conclusion, the fucanase from SW5, referred to as FcnA, should help in cracking the structure of the sulfated fucans of brown algae, a task which so far has proven difficult because of the lack of appropriate, specific fucan hydrolases. This fucan -(14) endohydrolase depolymerizes the sulfated fucan backbone, made of stretches of alternating -(14)-linked 2,3-disulfated fucose and -(13)-linked 2-sulfated fucose residues. FcnA thus provides another case study for understanding the structure–function relationships in the degradation of polysaccharides with high densities of sulfate-ester substituents. We indeed show here that, together with two distant homologous bacterial fucanases, FcnA delineates a novel structural family, thus opening a new opportunity to further decipher the structural bases of the degradation of sulfated polysaccharides.
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Preparation and analysis of sulfated fucan fractions
The preparation of the crude sulfated fucan fraction from the fucoid brown alga P. canaliculata, referred to as FS28, was described previously (Descamps et al., 2006
). Briefly, thalli were macerated overnight in 1 L of ethanol/formaldehyde/water (80:5:15; v/v) and exhausted by 2 L of ethanol/formaldehyde/water followed by 2 L of acetone. The resulting pellet was dried at 60°C and extracted twice for 3 h at 70°C by a 0.01 N HCl solution supplemented with 4% (w/v) calcium chloride. The extract was filtered and neutralized with ammonium carbonate, and fucans were precipitated with 2.5 volumes of ethanol, re-suspended in water, and freeze-dried. The resulting FS28 fraction containing 36% fucose (Descamps et al., 2006) was hygroscopic and displayed a brownish color, indicating the presence of polyphenols. To eliminate polyphenols, we further fractionated FS28 as follows. Aliquots (500 mg) were re-suspended in water (1%; w/v) and applied onto a Sephadex LH 20 column (GE Healthcare; total volume, 100 mL) equilibrated with water. The water-eluted fractions (total volume, 250 mL) were pooled, extensively dialyzed (cutoff 3500 Da) against distilled water, and freeze-dried. This white-colored sulfated fucan fraction contained 41% fucose. To eliminate LMW components, we ultrafiltrated this latter fraction against a 50-kDa cutoff membrane (Amicon). The retentate is a polymeric sulfated fucan fraction referred to as FC. To eliminate possible branching osidic residues (Chevolot et al., 1999) or some specific sulfate-ester substituents (Pomin et al., 2005), we dissolved FC in 0.5 M sulphuric acid (0.5%; w/v) and incubated at 40°C for 30 min. After neutralization with 4 M sodium hydroxide, the sample was dialyzed (cutoff 500 and 3500 Da) against water and freeze-dried. This sulfated fucan fraction is referred as FCH+.
Assays for fucanase activity
Attempts to quantitatively assay for fucanolytic activity using the release of sulfated fucan oligosaccharides by a conventional reducing sugar assay were unsuccessful (Nelson, 1944; Kidby and Davidson, 1973; Descamps et al., 2006). Sulfated fucan-degrading activity was thus monitored by a C-PAGE assay of the release of anionic oligosaccharides (Zablackis and Perez, 1990). Briefly, 100–250 µL of 0.1% (w/v) FS28 sulfated fucan in 20 mM Tris–HCl buffer (pH 7.5) was incubated with 1–5 µL of enzyme fraction for 1 h at room temperature. The products (5 µL) were electrophoresed through a 6% (m/v) stacking and a 27% running, 1-mm-thick polyacrylamide gel in 50 mM Tris–HCl, 2 mM EDTA buffer (pH 8.7), and stained with Alcian Blue followed by silver nitrate (Min and Cowman, 1986). Fucanolytic activity was detected by the occurrence of anionic oligosaccharide bands in the bottom part of the running gel. Product formation was also monitored as an increase of absorbance at 232 nm as a function of time. At room temperature, 5 µL of enzyme was added to a 1-mL cuvette containing 0.1% (w/v) FS28-sulfated fucan, and 20 mM Tris–HCl buffer (pH 7.5). The absorbance at 232 nm of the reaction mixture was followed for 30 min against a blank cuvette containing 0.1% (w/v) FS28-sulfated fucan, 20 mM Tris–HCl buffer (pH 7.5), and 5 µL of enzyme inactivated by heating at 100°C for 10 min.
Production and purification of fucanase FcnA
A 2-day-old culture in ZoBell-Fucan (ZF) medium of SW5 strain was inoculated (10 mL) in a 1-L Erlenmeyer flask containing 250 mL of ZF medium (Descamps et al., 2006) and cultured at 22°C, with a shaking speed of 200 rpm, for 1 day. Aliquots (40 mL) were then grown for 5 days in 5-L Erlenmeyer flasks in 6 x 1 L of the same medium under the same conditions, and bacterial growth was monitored by the absorbance at 600 nm. The culture supernatant was collected by centrifugation at 12,000 g for 20 min, filtered using a Pellicon system with 0.45-µm membrane (Millipore), and concentrated by ultrafiltration on a 30-kDa cutoff membrane (Millipore). The retentate (620 mL) was brought to 40% saturation with (NH4)2SO4, the suspension was centrifuged at 12,000 g for 15 min, and the resulting supernatant was brought to 60% (w/v) saturation with (NH4)2SO4. The precipitate was then collected by centrifugation at 12,000 g for 15 min and dissolved in 60 mL of 20 mM Tris–HCl buffer (pH 7.5), 5 mM MgCl2, 5 mM CaCl2, and 50 mM NaCl. This fraction was diluted 20-fold in a 20 mM Tris–HCl buffer (pH 7.5), resaturated at 40% with (NH4)2SO4, and deposited on a Phenyl Sepharose CL4B column (GE Healthcare) equilibrated with 100 mL of 20 mM Tris–HCl buffer (pH 7.5) and 40% (NH4)2SO4. The enzyme was eluted with the same buffer followed by a linear gradient of (NH4)2SO4 from 1.8 to 0 M (180 mL) in the same buffer. The active fractions (determined by their ability to release oligosaccharides from the FS28-sulfated fucan fraction as assayed by C-PAGE) were pooled and diluted 5-fold in a 20-mM Tris–HCl buffer (pH 7.5) containing 5 mM NaCl. The solution was applied onto a DEAE Sepharose CL6B column (GE Healthcare) equilibrated with 100 mL of 20 mM Tris–HCl buffer (pH 7.5, 5 mM NaCl). The column was washed with the same buffer (140 mL) and eluted with a linear gradient of 5 mM–1 M NaCl (210 mL). The active fractions were pooled (27 mL) and concentrated down to 1 mL by ultrafiltration on a 10-kDa cutoff membrane (Centricon, Millipore). The concentrate (1 mL) was applied onto a Superdex 200 (GE Healthcare) equilibrated with 50 mM sodium phosphate buffer (pH 7.0), 150 mM NaCl (100 mL), and the enzyme was eluted with the same buffer (30 mL). All purification steps were performed at 4°C. The apparent molecular weight of the purified enzyme was estimated by SDS–PAGE analysis, using a 12.5% polyacrylamide gel and a Phast-system (GE Healthcare), with standard proteins ranging from 14.5 to 200 kDa (broad range, Biorad, Marnes-la-Coquette, France). Protein contents were measured according to the Bradford method (Bradford, 1976) using a Biorad protein assay reagent with bovine serum albumin (Sigma, Lyon, France) as standard. Internal peptide microsequences were determined by Edman degradation at the Pasteur Institute (Paris, France).
Isolation and analysis of fucanase clones
Total DNA from SW5 strain was prepared as previously described (Barbeyron et al., 1984), cut by the restriction endonuclease Sau3AI, and fractionated on a sucrose gradient. DNA fragments of 4–10 kb were cloned at the BamHI site of plasmid vector pAT153 which was used to transform E. coli strain DH5. The genomic library contained 6000 clones. Using the microsequences obtained from internal peptides of the purified fucanase, we designed degenerated DNA primers and we obtained an internal DNA probe of 203 nt by PCR. The probe was labeled (kit Megaprime DNA labeling systems, GE Healthcare) and used to screen the library according to Sambrook and Russel (2001); then the clones of interest were mapped by restriction endonucleases. The probe was used for a Southern blot experiment carried out on genomic DNA, using various endonuclease combinations (HinDIII/BamHI, HinDIII, HinDIII/XcmI, EcoRI/NdeI, PstI/NdeI, EcoRI/PstI/NdeI, ClaI/NdeI, XcmI/NdeI, XcmI/PvuII, and EcoRI/SalI) (Sambrook and Russel, 2001). Sequencing was carried out by gene walking on both strands using a 3100 Genetic Analyser (Applied Biosystems, Courtaboeuf, France) with BigDye Terminator V3.0 chemistry (DNA sequencing kit, Applied Biosystems) and synthetic oligonucleotides as primers. Sequences were verified at least five times and most of them >10 times.
Sequence analysis
Nucleotide sequences were searched for ORFs, as well for putative ribosomal binding sequences, putative promoters, and putative transcription terminators hairpins via the mfold v.3.1 prediction server (Zuker et al., 1999). The signal peptide was predicted with SignalP V2.0 (Nielsen et al., 1999). The searches of homology were run with GENBANKnr and PAT via the NCBI web server using BLASTp and PSI-BLAST (Altschul et al., 1997). Evidence for specific motifs and domains was queried on the Pfam database (Bateman et al., 2004). The fucanase sequence was checked for repeats using the program RADAR (Heger and Holm, 2000).
Production of recombinant fucanase
The overexpression vector was constructed using GATEWAY cloning technology (Invitrogen, Cergy Pontoise, France), according to the supplier’s instructions. The coding region of SW5 fucanase, either as a whole or truncated after the He-PIg repeats or the N-terminal domain, was amplified by PCR from genomic DNA with high fidelity PLATINUM Pfx DNA polymerase (Invitrogen). PCR products were inserted into pDONR201 vector, the sequence was checked, and a valid clone was inserted into the pDEST17 vector with a N-terminal 6xHistidine tag. Plasmid vectors were used to transform BL21(DE3) pLysS E. coli strain (Novagen, Fontenay-Sous-Bois, France), and overexpression was carried out in the M9 medium supplemented with 2% casaminoacids, 100 µg mL–1 ampicillin, and 35 µg mL–1 chloramphenicol. Recombinant bacteria were grown in a Bioflow 3000 fermentor at 37°C (pH 7.2) and under-regulated oxygen concentration (70% of air). When cultures reached 0.8 OD600, expression was induced at 20°C with 1 mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG). Partially purified recombinant fucanases were produced as follows. After an overnight induction, bacterial cultures (500 mL) were centrifuged for 15 min at 10,000 g, and pellets were either frozen (–20°C) or resuspended in 50 mL of 20 mM Tris buffer (pH 7.5) containing 250 mM NaCl, 80 mM imidazol, and one tablet of Complete protease inhibitors (Roche Diagnostics, Meylan, France) and then disrupted by a French Press (4°C). The lysate was ultracentrifuged at 40,000 g for 1 h, and the supernatant was loaded on a Fast Flow Chelating Sepharose (GE Healthcare) column saturated with 100 mM NiSO4. The affinity chromatography column (V = 10 mL) was washed with lysis buffer (5 V) and eluted with an 80–300 mM imidazol gradient (12 V) and then by 1 M imidazol (2 V). Protein contents were estimated by SDS–PAGE, and aliquots of the recombinant enzyme were assayed for sulfated fucan-degrading activity by C-PAGE, as above.
SEC and 1H-NMR monitoring of the enzymatic degradation of sulfated fucan fractions
The enzymatic degradation of sulfated fucan was monitored by SEC as follows. Sulfated fucan aliquots (20–50 mg, 0.5–1% in water) were extensively digested with native or recombinant fucanase for 42 h at room temperature, then boiled for 10 min, and freeze-dried. Aliquots (10 mg) of the digestion products were resuspended (0.5–1%; w/v) in water and chromatographied with 50 mM ammonium carbonate using a Superdex 200 column whose outlet is connected to a Superdex peptide 10/300 GL column (GE Healthcare), equipped with refractometric detection. Liquid 1H-NMR spectra were recorded at 60°C on a Bruker DRX 500 Avance (Bruker, Wissembourg, France) at 500 MHz. Samples (10 mg) were previously exchanged twice in D2O (100%) before solubilization in 100% D2O.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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We are grateful to Drs Mirjam Czjzek, William Helbert, and Bernard Henrissat for helpful discussions. We thank also Miss Marion Guibet for her gift of purified
- and -carrageenan oligosaccharides. S.C. and V.D. were the recipients of PhD fellowships co-funded by the ANRT, whose help is gratefully acknowledged. |
Footnotes |
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The nucleotide sequence reported in this article has been submitted to the EMBL Nucleotide Sequence Database with accession number AJ877239.
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Abbreviations |
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C-PAGE, carbohydrate-polyacrylamide gel electrophoresis; HCA, hydrophobic cluster analysis; LMW, low molecular weight; ORF, open-reading frame; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography
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References |
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