Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Dec 2005; 71(12): 7670–7678.
PMCID: PMC1317386

Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

Abstract

Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

Xyloglucan is a widely distributed hemicellulose polysaccharide found in plant cell walls. It consists of a cellulose-like backbone of β-1,4-linked d-Glcp residues (β-1,4-glucan; p is a pyranose molecule) with side chains of α-d-Xylp residues attached at the C-6 position. Additional residues, Galp, Fucp, and Araf (where f is a furanose molecule), are also found in the side chain, but the branching patterns depend on the species of the plant. Single-letter nomenclature is used to simplify the naming of xyloglucan side-chain structures (5). For example, the letters G, X, and L refer to an unbranched Glc residue, an α-d-Xylp-(1→6)-β-d-Glcp segment, and a β-d-Galp-(1→2)-α-d-Xylp-(1→6)-β-d-Glcp segment, respectively. Structural studies suggest that most xyloglucans consist of repeating units of either XXXG (XXXG type) or XXGG (XXGG type) (26). XXXG type xyloglucans have three consecutive backbone residues substituted with Xyl residues and a fourth, unbranched Glc residue. XXGG type xyloglucans have two consecutively branched backbone residues and two unbranched backbone residues. Many endo-β-1,4-glucanases (cellulase [EC 3.2.1.4] and xyloglucanase [EC 3.2.1.151]) cleave the glycosidic bond of the unbranched Glc residues located in the backbone chain. Treatment of XXXG type xyloglucan with endoglucanase typically generates oligosaccharide subunits that have a tetrasaccharide backbone (XXXG, XLXG, XXLG, XLLG, etc.).

In the plant cell wall, xyloglucan associates with cellulose microfibrils via hydrogen bonds, forming a cellulose-xyloglucan network. During cell expansion and development, partial disassembly of the network is required (8, 9). Furthermore, xyloglucan metabolism is thought to have important roles in cell definition, cell expansion, and regulation of plant growth and development (1, 4, 17, 18, 31). For example, in the cell walls of the growing plant, xyloglucan oligosaccharides may provide positive- or negative-feedback control during cell elongation (25). In addition, the physiological effects of xyloglucan oligo- and polysaccharides have been shown in animals (16, 22-24, 27). These studies demonstrate the importance of analyzing the fine structure of xyloglucan in order to elucidate the relationship between its physiological function and its structure.

Glycosidases can be used as tools for analyzing the fine structure of complex carbohydrates. For example, isoprimeverose-producing oligoxyloglucan hydrolase (IPase) (EC 3.2.1.120), which cleaves xyloglucan oligosaccharide at the nonreducing end to produce an isoprimeverose [α-d-Xylp-(1→6)-β-d-Glcp-] residue, is a useful tool for analyzing xyloglucan and for preparing various xyloglucan oligosaccharide structures (9, 11-15, 33). An oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) (EC 3.2.1.150), which is a unique exoglucanase that cleaves xyloglucan oligosaccharides at their reducing ends (30), and a xyloglucan-specific endo-β-1,4-glucanase (xyloglucanase [XEG]) (29) from Geotrichum sp. strain M128, which we recently screened and cloned, have also been used for analyzing xyloglucan structure (11, 12). The screening and characterization of novel glycosidases are important for ensuring continued progress in glycotechnology. In this study, we report two novel xyloglucanases, XEG5 and XEG74, which belong to glycoside hydrolase families 5 and 74, respectively. Both enzymes were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21 and are active only toward xyloglucan and not toward carboxylmethyl cellulose (CMC), Avicel, barley β-1,3/1,4-glucan, or xylan.

MATERIALS AND METHODS

Purification of xyloglucanase.

The purification procedures are summarized in Fig. Fig.1.1. Paenibacillus sp. strain KM21 was isolated from soil based on its ability to degrade tamarind seed xyloglucan. The bacterium was cultured in 4 liters of a medium consisting of 0.8% Bacto peptone, 0.2% KH2PO4, 0.05% MgSO4, 0.1% NaCl, 0.1% Bacto yeast extract, and 0.8% tamarind seed xyloglucan (Dainippon Pharmaceutical, Osaka, Japan) at 30°C for 5 days with shaking. The cells were removed by centrifugation, and the supernatant was concentrated by ultrafiltration, dialyzed against 3 liters of water, and applied to a Poros 50 HQ anion-exchange column (2.6 by 20 cm; Perseptive Biosystems, MA) equilibrated with 200 ml of 100 mM HEPES-morpholineethanesulfonic acid-sodium acetate buffer (pH 7.5). The column was washed with 200 ml of the same buffer, and the flowthrough fraction was recovered. XEG5 did not bind to the Poros 50 HQ column and was in the flowthrough fraction, whereas XEG74 bound to the column and was eluted along a linear gradient between 0 and 0.6 M NaCl (300 ml in total). Each fraction was assayed for xyloglucan hydrolysis activity using the Nelson-Somogyi method, which measures the amount of reducing sugar (19-21).

FIG. 1.
Purification schemes for XEG5 and XEG74 from Paenibacillus sp. strain KM21.

The fractions containing xyloglucan hydrolysis activity (0.30 to 0.36 M NaCl) were pooled and dialyzed against 2 liters of 25 mM imidazole-HCl buffer (pH 7.4). The crude enzyme preparations were applied to a Poros 50 DEAE column (2.6 by 20 cm; Perseptive Biosystems) equilibrated with 200 ml of 25 mM imidazole-HCl buffer (pH 7.4). The column was washed with 200 ml of the same buffer and eluted with a linear gradient of 0 to 0.6 M NaCl (total, 300 ml). The fractions containing xyloglucanase activity (between 0.43 and 0.48 M NaCl) were combined and concentrated by ultrafiltration with Ultrafree-15 (Millipore). The concentrated enzyme preparation was applied to a Sephacryl S-300 HR gel filtration column (2.6 by 100 cm; Amersham Bioscience) equilibrated with 350 ml of 50 mM sodium acetate buffer (pH 6.0) containing 1.5 M NaCl and eluted with the same buffer. The fractions (5.5 ml/tube) with xyloglucan hydrolysis activity (fractions numbered 56 to 61) were pooled, concentrated with Ultrafree-15, and then subjected again to the same method of gel filtration chromatography. The fractions (5.5 ml/tube) with xyloglucan hydrolysis activity (fractions numbered 56 to 61) were pooled and dialyzed against 1 liter of 5 mM sodium acetate buffer (pH 6.0). The enzyme contained in these fractions migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. (Fig.2,2, lane 2).

FIG. 2.
SDS-PAGE of purified XEG5 and XEG74. Lane 1, purified XEG5; lane 2, purified XEG74. MW, molecular mass marker.

XEG5 was found in the flowthrough fraction from the Poros 50 HQ column. This fraction was dialyzed against 2 liters of 50 mM sodium acetate buffer (pH 5.0) and was then applied to a Poros 50 HS cation-exchange column (2.6 by 20 cm; Perseptive Biosystems) equilibrated with 200 ml of 50 mM sodium acetate buffer (pH 5.0). The column was washed with 200 ml of the same buffer, and fractions were eluted with a linear gradient of 0 to 0.6 M NaCl (total, 300 ml). The fractions containing xyloglucanase activity (0.21 to 0.36 M NaCl) were combined, concentrated with Ultrafree-15, applied to a Sephadex 75 gel filtration column (26 by 60 cm; Amersham Biosciences) equilibrated with 550 ml of 50 mM sodium acetate buffer (pH 6.0) containing 1.5 M NaCl, and then eluted with the same buffer. The fractions (3.5 ml/tube) containing xyloglucan hydrolysis activity (fractions numbered 59 to 61) were pooled and dialyzed against 1 liter of 5 mM sodium acetate buffer (pH 6.0). The enzyme contained in these fractions migrated as a single band on SDS-PAGE (Fig. (Fig.2,2, lane 1). The protein concentration was determined using BCA protein assay reagent (Pierce) with bovine serum albumin as the standard.

N-terminal and internal amino acid sequence analysis.

The purified proteins were separated by SDS-PAGE and transferred in transfer buffer (10 mM CAPS [cyclohexylaminopropanesulfonic acid], pH 11, 10% methanol) onto a polyvinylidene difluoride membrane filter (Millipore). The amino acid sequence was determined by automated sequential Edman degradations using a Procise 494 HT protein sequencing system (Applied Biosystems). To determine internal amino acid sequences, the purified protein was digested with lysyl endopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The resulting peptide fragments were fractionated by reverse-phase high-performance liquid chromatography (HPLC) on a TSK gel ODS-80Ts QA column (TOSOH Co., Tokyo, Japan), and their N-terminal amino acid sequences were analyzed.

Gene cloning and expression of XEG5.

Genomic DNA was prepared from Paenibacillus sp. strain KM21 using the FastDNA kit (BIO 101). Degenerate primers were designed based on internal amino acid sequences. The forward primers F1 and F2 and were based on peptides Xeg5-1 (AAYCAYATHGGNWSIGCICCNAA) and Xeg5-2 (ATHATGATHWSIGCNCAYTAYTA), respectively, and the reverse primers R1 and R2 were designed based on peptides Xeg5-3 (ACNGGRTAICCYTGNGTNACRAA) and Xeg5-4 (CCSTGSTGNGTIACNGTRTTRTT), respectively. PCR was conducted using genomic DNA as a template and either of the two primer pairs F1 and R1 or F2 and R2. The amplified fragments were subcloned into a T-overhang vector, pGEM-T Easy (Promega). Dideoxy double-stranded sequencing of the DNA insert was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions, and the expected amplified fragments were confirmed. To determine the complete nucleotide sequence, genomic DNA was digested with EcoRI, circularized by ligation, and subjected to inverse PCR using the gene-specific primers 5′-CCACCTTGCACGGTATTGTAGCCA-3′ and 5′-CCGGTTTATTGGGATAACGGCCAC-3′. The amplified fragment was sequenced using an ABI PRISM 310 Genetic Analyzer.

To express the mature region of XEG5 in Escherichia coli, the construct was first fused with a His6 tag at its C terminus. The DNA fragment encoding the mature region of XEG5 was amplified by PCR using the primers 5′-AAGCATCCATGGCAACTCCGATTACATCG-3′ and 5′-CGTATGAATCTCGAGTTGCATTCCTTGCATGATCGC-3′, which were designed based on the 5′ and 3′ sequences corresponding to the N- and C-terminal regions of the mature protein, respectively (the NcoI and XhoI sites are underlined and italicized, respectively). The amplified DNA was digested with NcoI and XhoI, subcloned into the pET-28a(+) expression vector (Novagen), digested with the same restriction enzymes, and transfected into E. coli BL21(DE3)(pLysS) (Novagen). The transfected cells were cultured, and expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h at 37°C. Soluble intracellular recombinant protein containing the His6 tag was extracted using the BugBuster protein extraction reagent (Novagen) and purified by metal chelate affinity chromatography using the HiTrap chelating column (Amersham Biosciences). The fractions containing XEG5 were pooled, dialyzed against 50 mM sodium acetate buffer (pH 5.0), loaded onto a RESOURCE S cation-exchange column (Amersham Biosciences) equilibrated with 50 mM sodium acetate buffer (pH 5.0), and eluted with a linear gradient of 0 to 0.5 M NaCl. Finally, XEG5 was subjected to gel filtration chromatography with a HiLoad 16/60 Superdex 200-pg column (Amersham Biosciences) in 25 mM imidazole-HCl buffer (pH 7.4).

Gene cloning and expression of XEG74.

Degenerate primers were designed from internal amino acid sequences: the forward primer from the N-terminal sequence TAYACNTGGAARAAYGTNGTNAC and the reverse primer from peptide Xeg74-2 (ACNGCCATYTCYTCNACNCC). A DNA fragment was amplified by PCR using these primers and genomic DNA as a template. The amplified fragment was subcloned into the T-overhang vector pGEM-T Easy (Promega) and was sequenced. To determine the complete nucleotide sequence, genomic DNA was digested with BstPI, circularized by ligation, and subjected to inverse PCR using the gene-specific primers 5′-ACGAACAATTTGACGAACTGGGAC-3′ and 5′-GTCCGTGCATAGATGAGGTCCTTC-3′. The amplified fragment was sequenced using an ABI PRISM 310 Genetic Analyzer.

To express the mature region of XEG74 in E. coli, the construct was first fused with a His6 tag at its C terminus. The DNA fragment encoding the mature region of XEG was amplified by PCR using the primers 5′-CTGAAAGGTCTCCCATGGCCCCGAGTGAACCGTATACGTG-3′ and 5′-GGATGACTCGAGGGGTTCAATCCCCCACTGCAGC-3′, designed from the 5′ and 3′ sequences corresponding to the N- and C-terminal regions of the mature protein, respectively (the BsaI, NcoI, and XhoI sites are underlined, double underlined, and italicized, respectively). As the XEG gene has an NcoI site, BsaI, which cleaves downstream of this recognition site, was used. Any of these restriction sites can be engineered into PCR primers such that NcoI-compatible overhangs are generated. The amplified DNA was digested with BsaI and XhoI, subcloned into the pET-28a(+) expression vector (Novagen), digested with NcoI and XhoI, and transfected into E. coli BL21-CodonPlus(DE3) RP (Stratagene). The transfected cells were cultured, and expression was induced with 0.1 mM IPTG for 4 h at 37°C. Soluble intracellular recombinant protein was extracted using the BugBuster protein extraction reagent (Novagen) and purified with a HiTrap chelating column (Amersham Biosciences). The fractions containing XEG74 were pooled, dialyzed against 25 mM imidazole-HCl buffer (pH 7.4), loaded onto a RESOURCE Q anion-exchange column (Amersham Biosciences) equilibrated with 25 mM imidazole-HCl buffer (pH 7.4), and then eluted with a linear gradient of 0 to 0.5 M NaCl. Finally, XEG74 was gel filtrated with a HiLoad 16/60 Superdex 200-pg column (Amersham Biosciences) in 25 mM imidazole-HCl buffer (pH 7.4).

Characterization of recombinant XEG5 and XEG74.

The temperature and pH effects of recombinant XEGs were analyzed. Xyloglucan hydrolase activity was assayed by measuring the reducing power using the Nelson-Somogyi method. The optimum temperature for enzyme activity (0.2 units/ml) was determined by incubation with tamarind seed xyloglucan (5 mg/ml) in 20 mM sodium phosphate buffer (pH 6.0) for 10 min at various temperatures. Thermostability was analyzed by incubating the enzyme without substrate in the same buffer for 10 min at various temperatures, and the remaining activity was then assayed by incubation with tamarind seed xyloglucan (5 mg/ml) at 45°C for 10 min. The optimum pH was determined by incubating the enzyme (0.2 units/ml) with tamarind seed xyloglucan (5 mg/ml) at 45°C for 10 min in McIlvaine buffer solutions (0.2 M disodium hydrogen phosphate and 0.1 M citric acid) that varied in pH (2.5 to 9.0). The pH stability was assayed by incubating the enzyme in the absence of substrate at 45°C for 30 min in the same buffer solutions. The buffer solutions were then adjusted to pH 6.0, and the remaining activity was assayed by incubation with tamarind seed xyloglucan (5 mg/ml) at 45°C for 10 min.

Kinetic constants were determined at xyloglucan concentrations ranging from 0.1 to 5 mg/ml using 2.5 μg enzyme/ml in 20 mM sodium phosphate buffer (pH 6.0). The bicinchoninate assay was used to quantify reducing sugars. The Michaelis constant (Km) and specific activity were calculated from the plot of initial reaction rates versus substrate concentration using Prism (Graphpad software).

The polysaccharide hydrolysis activity was assayed using the Nelson-Somogyi method (19-21). The hydrolyzing activities of recombinant XEGs toward tamarind seed xyloglucan, CMC (Nacalai Tesque, Inc., Kyoto, Japan), Avicel (Sigma), barley β-1,3/1,4-glucan (Sigma), and xylan (Sigma) were tested. Each polysaccharide (5 mg) was incubated at 45°C for 18 h in 1 ml of 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 U recombinant XEG; the amounts of the resulting reducing sugars were measured using the Nelson-Somogyi method. The final products were analyzed by normal-phase HPLC and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). HPLC was carried out with an Amide-80 normal-phase column (2.5 by 260 mm; TOSOH, Tokyo, Japan) using 57% acetonitrile (isocratic) at a flow rate of 0.6 ml/min. MALDI-TOF MS was performed with a Voyager mass spectrometer (Perseptive Biosystems) at an accelerating energy of 20 kV, in linear mode, and with positive-ion detection. The matrix was 2,5-dihydroxy-benzoic acid in 50% acetonitrile at a concentration of 10 mg/ml. XXXG served as an external calibration standard.

Preparation of xyloglucan oligosaccharides.

Various structures of xyloglucan oligosaccharides were prepared from tamarind seed xyloglucan, which is of the XXXG type and contains the subunits XXXG, XLXG, XXLG, and XLLG. Xyloglucan oligosaccharide, XLLGXLLG, and XXXGXXXG were obtained from partial endoglucanase digests of tamarind seed xyloglucan. A solution of tamarind seed xyloglucan (1%, wt/vol) was digested partially with Trichoderma viride cellulase (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the resulting oligosaccharides that have two subunits (e.g., XXXGXXXG, XXXGXXLG, XXLGXLLG, XLLGXLLG, etc.) were separated by gel filtration chromatography on a Bio Gel P2 column (Bio-Rad). Thereafter, two xyloglucan oligosaccharides, XXXGXXXG and XLLGXLLG, were separated on an Amide-80 normal-phase column.

The oligosaccharide XXXGXXXG was treated with Bacillus α-xylosidase (Seikagaku Co., Tokyo, Japan). This enzyme liberates xylose by hydrolysis of the α-xylosyl linkage at the nonreducing end of the xyloglucan oligosaccharide, and thus, the oligosaccharide product of α-xylosidase treatment of XXXGXXXG was expected to be GXXGXXXG. The product was separated by gel filtration chromatography, and purified oligosaccharide was monitored by normal-phase HPLC and MALDI-TOF MS.

For the preparation of XXGXXXG, GXXGXXXG was treated with a β-glucosidase from almond (Sigma). This enzyme liberates the glucose residue by hydrolyzing the β-glucosyl linkage at the nonreducing end of xyloglucan oligosaccharide, when the nonreducing end is an unbranched glucose residue. Thus, the oligosaccharide product of β-glucosidase treatment of GXXGXXXG was expected to be XXGXXXG. The product was separated by gel filtration chromatography, and purified oligosaccharide was monitored by HPLC and MALDI-TOF MS. Similarly, α-xylosidase and β-glucosidase were used to generate the oligosaccharides GXGXXXG (XXGXXXG treated with α-xylosidase), XGXXXG (GXGXXXG treated with β-glucosidase), and GGXXXG (XGXXXG treated with α-xylosidase).

XXXGXXXGG, XXXGG, and XXXGX were prepared by transglycosylation reactions using Trichoderma viride cellulase (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The transglycosylation reaction occurs when certain types of hydrolases are incubated with a high concentration of the acceptor, and this reaction was applied to xyloglucan (32). XXXGXXXG was incubated with cellulase and 20% glucose. The resulting products were XXXG, XXXGG, and XXXGXXXGG, which were separated by gel filtration chromatography. Likewise, XXXG was incubated with cellulase and 20% X (isoprimeverose) to produce XXXGX.

XXXGXX and XXXGXXX were prepared by incubating XXXGXXXG and XXXGXXXGG, respectively, with Geotrichum OXG-RCBH, which releases two Glc residue segments from the reducing end of the xyloglucan oligosaccharide main chain (30).

Substrate specificity analysis of XEGs using various xyloglucan oligosaccharides.

Substrate specificity was analyzed using well-defined xyloglucan oligosaccharide structures. Each oligosaccharide (0.2 mg) was incubated in 20 μl of 20 mM sodium phosphate buffer (pH 6.0) containing 0.2 U recombinant XEG at 45°C. After 18 h, the resulting products were analyzed by normal-phase HPLC and MALDI-TOF MS.

Viscosimetric assay.

Viscosimetric assays were carried out by monitoring the flow time of 0.8% xyloglucan in 20 mM sodium phosphate buffer (pH 6.0) incubated with XEG at different times. The flow time of the reaction mixture was determined in an Oswald viscometer at room temperature, and the reducing-sugar content was determined by bicinchoninate assay. After various incubation times, the specific viscosity was calculated as (TT0)/T0, where T0 is the flow time measured for the buffer and T is the flow time of the reaction mixture with the enzyme.

Analysis of xyloglucan hydrolysis products by gel filtration chromatography.

Xyloglucan (0.8%) in 20 mM sodium phosphate buffer (pH 6.0) was incubated with XEG. After various incubation times, the reaction solution was applied to a Superdex Peptide 10/300 GL (Amersham Biosciences) gel filtration column, and the degradation products were analyzed.

Nucleotide sequence accession numbers.

The nucleotide sequences of XEG5 and XEG74 have been deposited in the DDBJ/EMBL/GenBank database under accession numbers AB212089 and AB212090, respectively.

RESULTS AND DISCUSSION

Purification of XEGs.

The purification schemes are summarized in Fig. Fig.1.1. The supernatant obtained from a culture of Paenibacillus sp. strain KM21 served as the starting material and was concentrated by ultrafiltration. Two forms of XEG were detected; these forms were well separated chromatographically on a Poros 50 HQ column by binding to or flowing through the column. XEG74 bound to Poros 50 HQ and then to Poros 50 DEAE; following chromatography on a Sephacryl S-300 HR column, the purified enzyme was subjected to SDS-PAGE, revealing a single band of about 105 kDa (Fig. (Fig.2,2, lane 2). XEG5 flowed through the Poros 50 HQ column but bound to Poros 50 HS. The enzyme was chromatographed on Sephadex 75 and then subjected to SDS-PAGE, migrating as a single band of about 40 kDa (Fig. (Fig.2,2, lane 1). XEG5 and XEG74 were active only toward xyloglucan and not toward the polysaccharides CMC, Avicel, barley β-1,3/1,4-glucan, or xylan.

N-terminal and internal amino acid sequences.

Purified XEGs were analyzed to determine their N-terminal amino acid sequences. The sequence of XEG5 was identified as ATPITSDFRSLQAXQIVSEM, and that of XEG74 was identified as APSEPYTWKNVVTGAGGGFV. Internal amino acid sequences were obtained by digesting XEGs with lysyl endopeptidase and separating the resulting peptide fragments using reverse-phase HPLC. The N-terminal amino acid sequences of these fragments were shown to be as follows: peptide Xeg5-1, IPVSYLNHIGSAPNY; peptide Xeg5-2, IMISAHYYSPXDFAG; peptide Xeg5-3, FVTQGYPVVLGEFGS; peptide Xeg5-4, SNNTVTQQGIINAIM; peptide Xeg74-1, FRYTQNISAAPWLTF; peptide Xeg74-2, GVEEMAVLDLVSPPS.

Gene cloning of XEG5 and XEG74.

PCR was carried out using genomic DNA from Paenibacillus sp. strain KM21 as a template with degenerate primers designed from the amino acid sequences of purified XEG5 and XEG74. The amplified DNA fragments were subcloned and sequenced. Inverse PCR was used to determine the complete DNA nucleotide sequences, followed by cloning of the full-length DNA that encoded XEG5 and XEG74.

The sequence of XEG5 contains a 1,215-bp open reading frame that encodes a putative protein of 405 amino acids. The deduced amino acid sequence matched the partial amino acid sequences obtained from the purified protein. The N-terminal sequence of the mature protein started at the 33rd residue from the first Met, indicating that 32 amino acids were cleaved from the N terminus of the mature form. These 32 amino acids are probably a signal sequence. N-terminal proteolytic cleavage resulted in a 373-amino-acid protein with an average molecular mass of 41,375 Da as deduced from the amino acid sequence. This molecular mass is similar that of the native enzyme as determined by SDS-PAGE (Fig. (Fig.2,2, lane 1).

The deduced amino acid sequence of XEG5 was aligned using a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/). The alignment revealed that the catalytic domain sequence of XEG5, which follows the N-terminal signal peptide, was highly similar to the sequences of enzymes belonging to the glycoside hydrolase family 5 (GH5), as classified according to the CAZy database (http://afmb.cnrs-mrs.fr/CAZY/) (Fig. (Fig.3).3). Other members of GH5 include chitosanase (EC 3.2.1.132), β-mannosidase (EC 3.2.1.25), cellulase (EC 3.2.1.4), glucan 1,3-β-glucosidase (EC 3.2.1.58), licheninase (EC 3.2.1.73), glucan endo-1,6-β-glucosidase (EC 3.2.1.75), mannan endo-1,4-β-mannosidase (EC 3.2.1.78), endo-1,4-β-xylanase (EC 3.2.1.8), cellulose 1,4-β-cellobiosidase (EC 3.2.1.91), endo-1,6-β-galactanase, and β-1,3-mannanase. However, XEG5 is the first xyloglucan-specific endoglucanase belonging to GH5. Interestingly, the endoglucanase CelA from Bacillus sp. strain BP-23, which has 80% amino acid sequence identity to the mature region of XEG5, was previously reported to be a cellulase (2). As the activity of Bacillus CelA toward xyloglucan was not tested, it remains possible that this enzyme has high activity toward this substrate; however, XEG5 does not digest cellulose. The slight difference in amino acid sequence between CelA and XEG5 may therefore determine substrate specificity, i.e., active or inactive toward cellulose.

FIG. 3.
Schematic illustrating the modular architecture of XEG5 and XEG74. XEG5 contains an N-terminal signal sequence (striped) followed by a GH5 catalytic domain. XEG74 contains an N-terminal signal sequence (striped) followed by a GH74 catalytic domain, an ...

The DNA sequence of XEG74 contains a 3,081-bp open reading frame that encodes a putative protein of 1,027 amino acids. The deduced amino acid sequence matched the partial amino acid sequences obtained from the purified protein. The N-terminal amino acid sequence of the mature protein started at the 33rd residue from the first Met, indicating that 32 amino acids were cleaved from the N terminus of the mature form. These 32 amino acids are probably a signal sequence. N-terminal proteolytic cleavage resulted in a 995-amino-acid protein with an average molecular mass of 105,867 Da as deduced from the amino acid sequence. This molecular mass is similar to that of the native enzyme as determined by SDS-PAGE (Fig. (Fig.2,2, lane 2).

The deduced amino acid sequences were aligned using a BLAST search and Pfam (http://www.sanger.ac.uk/Software/Pfam/). The results revealed that XEG74 had an N-terminal signal peptide followed by a catalytic domain that belonged to the glycoside hydrolase family 74 (GH74), an X2 module, and a carbohydrate-binding module family 3 (CBM3) region (Fig. (Fig.3).3). The X2 modules are found in some glycosidases and cellulosomes, but their functions are unknown. CBM3s have been identified in bacterial enzymes, and cellulose-binding ability has been demonstrated in many cases. In this study, the full-length XEG74 bound to crystalline cellulose (Avicel), while XEG74 without CBM3 did not (data not shown). These results suggest that the CBM3 region of XEG74 has the ability to bind cellulose, as do the CBM3 regions of other family members. Recently, other xyloglucanases belonging to GH74 have been reported. Aspergillus niger EglC is an endoglucanase with strong activity toward xyloglucan and weaker activities toward CMC and β-glucan (7). Thermobifida fusca Xeg74 and Geotrichum XEG are endoglucanases that specifically act on xyloglucan (10, 29), as was shown for XEG74. By contrast, Geotrichum OXG-RCBH and Trichoderma reesei Cel74 are exoglucanases that are active toward xyloglucan. Cel74 cleaves tamarind seed xyloglucan to XXXG, XXLG, XLXG, and XLLG by an exo mode of action (6), whereas OXG-RCBH is a unique enzyme that releases two glucosyl residues from the reducing end of the main chain of xyloglucan oligosaccharide (30). The crystal structure of OXG-RCBH has recently been resolved (28). The enzyme has an active cleft, and one of its sites is closed by a loop region that has not been observed in other GH74 enzymes with an endo mode of action. This loop prevents binding to the middle of the xyloglucan chain and thus bestows exo activity. By contrast, XEG74 does not have the loop region, suggesting that it has endo activity.

Expression and characterization of recombinant XEGs.

The mature regions of XEG5 and XEG74 were expressed in E. coli cells, and recombinant XEGs fused with a C-terminal His tag were found in the BugBuster soluble fraction, as were their enzymatic activities. These results showed that recombinant XEGs could be expressed in a soluble, enzymatically active form. The recombinant XEGs were purified using the chromatography procedures described in Materials and Methods, and the effects of pH and temperature on the enzymes were analyzed. The optimum pH of XEG5 was 5.5 to 6.5, and the optimum temperature was 50 to 55°C. XEG5 was stable between pH 5.0 and 8.0 at 45°C. Thermostability was analyzed by incubating the enzyme at various temperatures for 10 min. More than 90% activity remained at 55°C. The optimum pH and temperature of XEG74 were pH 6.0 to 6.5 and 60 to 70°C, and the enzyme was stable between pH 5.0 and 7.5 and up to 55°C.

Kinetic constants were determined for various concentrations of xyloglucan. The Km of XEG5 was 2.0 mg/ml, the specific activity was 18.4 U/mg protein, and the kcat was 12.7 s−1. One unit was defined as the amount of enzyme that released 1 μmol of glucose equivalents as reducing sugars from xyloglucan per minute. The Km, specific activity, and kcat of XEG74 were 1.2 mg/ml, 44.0 U/mg protein, and 77.6 s−1, respectively. There were no significant differences in these constants between full-length XEG74 and a truncated enzyme that contained only the catalytic domain, i.e., without X2 and the CBM3 region (Km, specific activity, and kcat were 1.0 mg/ml, 49.8 U/mg protein, and 65.6 s−1, respectively). The differences in the kcat values between XEG5 and XEG74 may be associated with the different reaction mechanisms of the two enzymes.

Substrate specificities of XEGs.

The hydrolyzing activities of recombinant XEGs towards tamarind seed xyloglucan, CMC, Avicel, barley β-1,3/1,4-glucan, and xylan were tested. XEG5 and XEG74 were active only toward xyloglucan and not toward other polysaccharides. The final products were analyzed by normal-phase HPLC and MALDI-TOF MS. Figure Figure44 shows the digestion products of tamarind seed xyloglucan by XEG74. These consisted of XXXG, XLXG, XXLG, and XLLG. XEG5 produced the same products as XEG74 (data not shown). There were no peaks corresponding to molecules larger than XLLG, which indicated that, at least under these conditions, XEG5 and XEG74 had no transglycosylating activity (data not shown).

FIG. 4.
Analysis of the final products resulting from the complete digestion of tamarind seed xyloglucan by XEG74. The reaction products were analyzed by HPLC (A) and MALDI-TOF MS (B).

The results suggest that both enzymes cleave the glucosidic bonds of unbranched Glc residues, which is a reaction similar to those of other known β-1,4-glucanases. In addition, XEG5 and XEG74 cleaved XLLGXLLG to XLLG (data not shown). Interestingly, although XEG5 cleaved XXXGXXXG to XXXG (Fig. (Fig.5B),5B), XEG74 produced not only XXXG but also two different peaks, referred to as product A and product B (Fig. (Fig.5C).5C). These peaks were collected independently, and the fractionated products were analyzed by MALDI-TOF MS, which showed that the molecular mass of product A corresponded to XXX (Fig. (Fig.5D)5D) and that of product B corresponded to GXXXG (Fig. (Fig.5E).5E). Products A and B were also treated with Aspergillus oryzae IPase (14) and β-glucosidase. After digestion with IPase, product A yielded only isoprimeverose, while product B was not cleaved (data not shown). By contrast, after digestion with β-glucosidase, product A was not cleaved, whereas product B yielded glucose and XXXG (data not shown). These results demonstrate that XEG74 produced not only XXXG but also XXX and GXXXG from XXXGXXXG. The cleavage ratio, calculated by quantifying the areas of each peak, showed that XXXG accounted for 69% of the XEG74 products and that XXX and GXXXG accounted for 31%. Thus, XEG74 is the first endoglucanase that cleaves the glucosidic bond of branched Glc residues (glucosidic bond between -X-G-) of XXXGXXXG. By contrast, the only digestion product from XLLGXLLG was XLLG, which indicated that XEG74 cleaved only the glucosidic bonds of unbranched Glc residues (glucosidic bond between -G-X-). This result indicated that the cleavage of the glucosidic bond of the branched Glc residues was inhibited by Gal residues attached to Xyl residues. This would appear to explain why XEG74 cleaved the glucosidic bond of the unbranched Glc residues of tamarind seed xyloglucan polysaccharide to produce XXXG, XXLG, XLXG, and XLLG (Fig. (Fig.4).4). In addition, it is possible that the degree of polymerization of the substrate affected the enzymatic activity. To confirm this, experiments with various additional substrates, such as XXXGXXXGXXXG, XXXGXXXGXXXGXXXG, and XXXGXXXGXXXGXXXG, are required. We are in the process of preparing and analyzing the enzymatic activity with these substrates.

FIG. 5.
HPLC and MALDI-TOF MS analysis of the digestion products of the xyloglucan oligosaccharide XXXGXXXG. (A to C) HPLC profiles of XXXGXXXG (A) and the digestion products by XEG5 (B) and XEG74 (C). (D to F) MALDI-TOF MS analysis of the digestion products ...

An enzyme from the same family (GH74), Geotrichum XEG, cleaves XXXGXXXG to XXXG and does not produce XXX or GXXXG (29). As no three-dimensional structures are available for XEG74 or Geotrichum XEG, structural analyses are required to understand the mechanism of substrate specificity for these enzymes. We are currently analyzing the crystal structures of both enzymes.

XEG74 substrate specificity was further analyzed using various xyloglucan oligosaccharide structures (Fig. (Fig.6).6). XEG74 digested XGXXXG, GGXXXG, and XXXGX at the second and third positions from their nonreducing ends, while other xyloglucan oligosaccharides were digested at the third and fourth positions. The predominant cleavage positions depended on the structure of the oligosaccharide: XXGXXXG, XXXGXX, GGXXXG, and XXXGX at the third position; XXXGXXXG and GXXGXXXG at the fourth position; and GXGXXXG, XGXXXG, and XXXGXXX in equal amounts at the third and fourth positions.

FIG. 6.
Substrate specificity of XEG74 toward xyloglucan oligosaccharides. Xyloglucan oligosaccharides were incubated with recombinant XEG74. The resulting digestion products were quantified and analyzed by HPLC and MALDI-TOF MS. The cleavage sites and ratios ...

Viscosimetric assay and gel filtration chromatography of the degradation products.

The hydrolytic activities of XEG5 and XEG74 were analyzed by studying the decrease in xyloglucan viscosity compared to the release of reducing sugars. Viscosimetric assays were carried out by monitoring the flow time of 0.8% xyloglucan. After different incubation times, specific viscosity was determined. Xyloglucan was degraded concomitant with a decrease in viscosity, revealing that both XEG5 and XEG74 have endo modes of action. However, XEG5 reduced the viscosity of xyloglucan more rapidly than did XEG74 (Fig. (Fig.77).

FIG. 7.
Viscosimetric analysis. Tamarind seed xyloglucan was incubated with XEG5 or XEG74. After various incubation times, the specific viscosity was calculated and the hydrolysis ratio was determined by measuring the reducing power. The reducing power obtained ...

Xyloglucan was incubated with the two XEGs, and after the different incubation times, the degradation products were analyzed by gel filtration chromatography. In the case of XEG5, the molecular masses decreased rapidly, even at the initial stage of the reaction, suggesting that the degradation process involved random β-1,4-linkage hydrolysis along the xyloglucan polymer chain (Fig. (Fig.8A).8A). Thus, XEG5 is a typical endo-type enzyme. In the case of XEG74, although the molecular masses decreased slowly, the final degradation products (XXXG, XLXG, XXLG, and XLLG) were observed as early as during the initial stage of the reaction (Fig. (Fig.8B).8B). This type of reaction profile is found in exo-type or processive-type enzymes, suggesting that XEG74 has dual exo-mode and endo-mode activities, or an endo-processive mode of action, toward xyloglucan.

FIG. 8.
Analysis of xyloglucan hydrolysis products by gel filtration chromatography. Tamarind seed xyloglucan was incubated with XEG5 (A) or XEG74 (B) for various incubation times, and the reaction products were applied to a gel filtration column.

XEG74 does not contain the loop region that blocks one of the sites of the active cleft, as is found in OXG-RCBH, providing further evidence of the enzyme's endo activity. Interestingly, the GH74 xyloglucanase, Geotrichum XEG, is a typical endo-type enzyme that randomly cleaves xyloglucan polymer (data not shown) and in this way differs from XEG74. We are currently analyzing the crystal structures of XEG74 and Geotrichum XEG. These studies should provide insight into the mechanisms of their different activities.

Conclusion.

In this study, two xyloglucanases, XEG5 and XEG75, were isolated from Paenibacillus sp. strain KM21. The genes that encoded these enzymes were cloned and expressed in E. coli. Both native and recombinant XEGs had similar enzymatic properties (pH and temperature effects, kinetic parameters, and substrate specificity) (data not shown).

XEG5 is the first xyloglucan-specific endoglucanase belonging to the GH5 family. The deduced amino acid sequence shows high similarity with that of the Bacillus sp. strain BP-23 cellulase, CelA (2). Thus, it is possible that CelA has high activity toward xyloglucan, although this was not tested. It may also be that many other “cellulases” have greater activity toward xyloglucan than toward cellulose, but most cellulases have not been tested for xyloglucan-hydrolyzing activity. For example, A. niger EglC was the first functionally analyzed GH74 enzyme. It has strong activity for xyloglucan and weaker activity for CMC (7). If EglC had not been tested for xyloglucan hydrolysis activity, the enzyme would have been reported as a “cellulase,” and we perhaps would not have realized that GH74 enzymes have strong xyloglucan hydrolysis activities.

Xyloglucan-specific glycosidases with high substrate specificity, such as Geotrichum XEG and OXG-RCBH, are useful tools for analyzing xyloglucan structure (11, 12). As XEG74 has low substrate specificity, it is not appropriate for analyzing xyloglucans; however, it can be used to prepare various oligosaccharide structures, some of which are difficult to prepare without this enzyme. In this study, the oligosaccharides GXXXG, XXXG, GXXX, GXXG, XXGX, GXGX, XXX, XXG, XGX, GGX, GXX, GXG, XX, XG, GX, and GG were generated by XEG74. In addition, it may be possible to generate new, unique oligosaccharides by modifying the substrate specificity of XEG74 through genetic engineering. The availability of several types of glycosidases enables the preparation of a wide range of oligosaccharide structures that can be used to identify the substrate specificities of xyloglucan hydrolases, as in this study, as well as to elucidate the physiological functions of xyloglucan oligosaccharides in plants and animals. Such studies may result in the development of novel applications for these molecules.

REFERENCES

1. Augur, C., L. Yu, K. Sakai, T. Ogawa, P. Sinay, A. G. Darvill, and P. Albersheim. 1992. Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth. Plant Physiol. 99:180-185. [PMC free article] [PubMed]
2. Blanco, A., P. Díaz, J. Martínez, T. Vidal, A. L. Torres, and F. I. J. Pastor. 1998. Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw. Appl. Microbiol. Biotechnol. 50:48-54. [PubMed]
3. Reference deleted.
4. Fry, S. C. 1989. The structure and functions of xyloglucan. J. Exp. Bot. 40:1-11.
5. Fry, S. C., W. S. York, P. Albersheim, A. Darvill, T. Hayashi, J. P. Joseleau, Y. Kato, E. P. Lorences, G. A. Maclachlan, M. McNeil, A. J. Mort, J. S. G. Reid, H. U. Seitz, R. R. Selvendran, A. G. J. Voragen, and A. R. White. 1993. An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol. Plant. 89:1-3.
6. Grishutin, S. G., A. V. Gusakov, A. V. Markov, B. B. Ustinov, M. V. Semenova, and A. P. Sinitsyn. 2004. Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim. Biophys. Acta 1674:268-281. [PubMed]
7. Hasper, A. A., E. Dekkers, M. van Mil, P. J. I. van de Vondervoort, and L. H. de Graaff. 2002. EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan. Appl. Environ. Microbiol. 68:1556-1560. [PMC free article] [PubMed]
8. Hayashi, T. 1989. Xyloglucans in the primary-cell wall. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:139-168.
9. Hayashi, T., K. Ogawa, and Y. Mitsuishi. 1994. Characterization of the adsorption of xyloglucan to cellulose. Plant Cell Physiol. 35:1199-1205.
10. Irwin, D. C., M. Cheng, B. Xiang, J. K. C. Rose, and D. B. Wilson. 2003. Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca. Eur. J. Biochem. 270:3083-3091. [PubMed]
11. Ito, S., Y. Mitsuishi, T. Okuno, and Y. Kato. 2004. Changes in the structure of xyloglucan of apple fruit during development. J. Jpn. Soc. Hort. Sci. 73:51-56.
12. Kato, Y., S. Ito, and Y. Mitsuishi. 2004. Further structural study of the barley and bamboo shoot xyloglucans by xyloglucanase. J. Appl. Glycosci. 51:327-333.
13. Kato, Y., T. Konishi, Y. Hidano, and Y. Mitsuishi. 2004. Structural analysis of the eggplant xyloglucan by xyloglucanase. J. Appl. Glycosci. 51:93-99.
14. Kato, Y., J. Matsushita, T. Kubodera, and K. Matsuda. 1985. A novel enzyme producing isoprimeverose from oligoxyloglucans of Aspergillus oryzae. J. Biochem. (Tokyo) 97:801-810. [PubMed]
15. Konishi, T., Y. Mitsuishi, and Y. Kato. 1998. Analysis of the oligosaccharide units of xyloglucans by digestion with isoprimeverose-producing oligoxyloglucan hydrolase followed by anion-exchange chromatography. Biosci. Biotechnol. Biochem. 62:2421-2424. [PubMed]
16. Mastromarino, P., R. Petruzziello, S. Macchia, S. Rieti, R. Nicoletti, and N. Orsi. 1997. Antiviral activity of natural and semisynthetic polysaccharides on the early steps of rubella virus infection. J. Antimicrob. Chemother. 39:339-345. [PubMed]
17. McDougall, G. J., and S. C. Fry. 1988. Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan. Planta 175:412-416. [PubMed]
18. McDougall, G. J., and S. C. Fry. 1990. xyloglucan oligosaccharides promote growth and activate cellulase—evidence for a role of cellulase in cell expansion. Plant Physiol. 93:1042-1048. [PMC free article] [PubMed]
19. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380.
20. Somogyi, M. 1945. A new reagent for determination of sugars. J. Biol. Chem. 160:61-68.
21. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19-23. [PubMed]
22. Sone, Y., C. Makino, and A. Misaki. 1992. Inhibitory effect of oligosaccharides derived from plant xyloglucan on intestinal glucose-absorption in rat. J. Nutr. Sci. Vitaminol. 38:391-395. [PubMed]
23. Strickland, F. M., A. Darvill, P. Albersheim, S. Eberhard, M. Pauly, and R. P. Pelley. 1999. Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides. Photochem. Photobiol. 69:141-147. [PubMed]
24. Strickland, F. M., J. M. Kuchel, and G. M. Halliday. 2004. Natural products as aids for protecting the skin's immune system against UV damage. Cutis 74:24-28. [PubMed]
25. Takeda, T., Y. Furuta, T. Awano, K. Mizuno, Y. Mitsuishi, and T. Hayashi. 2002. Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments. Proc. Natl. Acad. Sci. USA 99:9055-9060. [PMC free article] [PubMed]
26. Vincken, J. P., W. S. York, G. Beldman, and A. G. J. Voragen. 1997. Two general branching patterns of xyloglucan, XXXG and XXGG. Plant Physiol. 114:9-13. [PMC free article] [PubMed]
27. Yamatoya, K., M. Shirakawa, K. Kuwano, J. Suzuki, and T. Mitamura. 1996. Effects of hydrolyzed xyloglucan on lipid metabolism in rats. Food Hydrocoll. 10:369-372.
28. Yaoi, K., H. Kondo, N. Noro, M. Suzuki, S. Tsuda, and Y. Mitsuishi. 2004. Tandem repeat of a seven-bladed β-propeller domain in oligoxyloglucan reducing-end-specific cellobiohydrolase. Structure 12:1209-1217. [PubMed]
29. Yaoi, K., and Y. Mitsuishi. 2004. Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128. FEBS Lett. 560:45-50. [PubMed]
30. Yaoi, K., and Y. Mitsuishi. 2002. Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. J. Biol. Chem. 277:48276-48281. [PubMed]
31. York, W. S., A. G. Darvill, and P. Albersheim. 1984. Inhibition of 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments by a xyloglucan oligosaccharide. Plant Physiol. 75:295-297. [PMC free article] [PubMed]
32. York, W. S., and R. Hawkins. 2000. Preparation of oligomeric β-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a Trichoderma reesei cellulase. Glycobiology 10:193-201. [PubMed]
33. York, W. S., H. van Halbeek, A. G. Darvill, and P. Albersheim. 1990. Structural analysis of xyloglucan oligosaccharides by 1H-NMR spectroscopy and fast-atom-bombardment mass spectrometry. Carbohydr. Res. 200:9-31. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...