A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides

Ciocchini et al. 10.1073/pnas.0708025104.

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SI Figure 7
SI Figure 8
SI Text
SI Figure 9
SI Figure 10




SI Figure 7

Fig. 7. Western blot analysis of Cgs truncated mutants. Total membrane proteins (100 mg) of A1045 strains harboring the indicated plasmid were subjected to SDS-/PAGE (1), followed by electrophoretic transfer to a nitrocellulose membrane (Inmobilon-NC; Millipore). Immunoblotting was performed as previously described (2) with a specific polyclonal antiserum against Cgs residues 540-800 (3). Bound antibody was visualized by using horseradish peroxidase-conjugated goat anti-mouse Ig (DAKO) and Supersignal West Pico chemiluminescent substrate detection reagents (Pierce Chemical) according to the manufacturer's instructions. pBA24, plasmid expressing wild-type Cgs. The position of the molecular mass standard (in kDa) is indicated on the left. The arrows on the right indicate the position of the Cgs protein.

1. Laemmli UK (1970) Nature (London) 227:680-685.

2. Burnett WN (1981) Ann Biochem 112:195-203.

3. Ciocchini AE, Roset MS, Inon de Iannino N, Ugalde RA (2004) J Bacteriol 186:7205-13.





SI Figure 8

Fig. 8. Isolation and purification of CbG. Cells from stationary phase cultures were harvested, and cell pellets were extracted with ethanol (70% ethanol, 1 h at 37°C). The ethanolic extract was centrifuged, and the supernatant was concentrated and subjected to gel filtration on Bio-Gel P4 columns. The truncated forms Cgs-1587stop and Cgs-2079stop and the pentapeptide insertion mutant Cgs-2319 produced CbG with high DP (A). Fractions 15-21 of Bio-Gel P4 elution profiles were pooled, concentrated, and analyzed by TLC. CbG that eluted from the column between these fractions correspond to CbG with reduced TLC mobility, that is, with high DP (B). Fractions 15-35 corresponding to total CbG were pooled, concentrated, and applied to a DEAE-Sephadex A-25 column. Fractions corresponding to neutral-CbG were analyzed by TLC (C). pBA24, plasmid expressing wild-type Cgs.





SI Figure 9

Fig. 9. Structure-based sequence alignment with B. abortus Cgs C-terminal region and the enzyme chitobiose phosphorylase (ChBP, BAC87867) from Vibrio proteolyticus (overall identity, 27.1%). The alignment was generated by Phyre (Structural Bioinformatics group, Imperial College, London). The predicted secondary structure for Cgs and the known secondary structure for ChBP are indicted (blue and red arrows indicate a-helices and b-strands, respectively). The alignment was edited with the Jalview 2.2 program (1). The catalytic residue of ChBP and the corresponding residue of Cgs are indicated by a red rectangle. The "fixer" residue (see text) of ChBP and the corresponding residue of Cgs are indicated by a blue rectangle. Residues involved in the recognition of GlcNAc in ChBP are indicated by orange rectangles. Residues involved in the recognition of phosphate in ChBP are indicated by green rectangles. The N-terminal domain, the helical linker region, the a-helical barrel domain, and the C-terminal domain comprising the structure of ChBP are indicated (2).

1. Clamp M, Cuff J, Searle SM, Barton GJ (2004) Bioinformatics 20:426-7.

2. Hidaka M, Honda Y, Kitaoka M, Nirasawa S, Hayashi K, Wakagi T, Shoun H, Fushinobu S (2004) Structure 12:937-47.





SI Figure 10

Fig. 10. SDS/PAGE analysis of recombinant C-terminal domain of Cgs. Lanes 1-3, crude extract from Escherichia coli BL21-CodonPlus(DE3)-RP cells harboring pCT, pCT-D2420A, and pCT-D2555A, respectively (40 mg). Lanes 3-6, purified recombinant Cgs-CT, Cgs-CT-D2420A, and Cgs-CT-D2555A, respectively (2 mg). The position of the molecular mass standard (in kDa) is indicated on the left. The arrow on the right indicates the position of the recombinant C-terminal Cgs proteins.





SI Text

SI Results

Sequence Analysis of Cgs. B. abortus Cgs displays high sequence similarity with the cyclic glucan synthases of Agrobacterium tumefaciens (ChvB, 53% of identity) and Sinorhizobium meliloti (NdvB, 54% of identity), as well as with the other cyclic glucan synthases sequenced so far (1). Sequence analysis revealed that Cgs is a modular protein with two defined regions: the N-terminal region (from amino acids 1 to 1544), which contains the GT-84 domain, and the C-terminal region (from amino acids 1545 to 2867), which displays significant sequence similarity to members of the glycoside phosphorylase family 94 (GH-94) (Fig. 3 A and B). GH-94 groups phosphorolytic enzymes, such as cellobiose phosphorylases (CBP), chitobiose phosphorylases (ChBP), and cellodextrin phosphorylases (CDP). These enzymes, using inorganic phosphate as the nucleophilic substrate, catalyze the phosphorolysis of b-glycosidic bonds generating glycosyl-phosphates with inversion of the anomeric configuration. A distinctive feature of cyclic glucan synthases is that the region corresponding to the N-terminal region of GH-94 glycoside phosphorylases is duplicated, and these two regions are connected by a linker region of 255 amino acid residues (Fig. 3A). A putative carbohydrate-binding domain (CBM-X) and a GH-94-associated family domain (GH-94 AF) were identified in each of these regions by NCBI Conserved Domain search (Fig. 3A) (2).

In addition, the analysis of Cgs C-terminal region (residues 1545-2867) by the Protein Homology/analogY Recognition Engine (Phyre) program (Structural Bioinformatics group, Imperial College, London) clearly revealed a predicted structure similar to that of chitobiose phosphorylase (ChBP) from Vibrio proteolyticus (estimated precision, 100%), a GH-94 member whose crystal structure has been resolved (3). Vibrio proteolyticus ChBP catalyzes the reversible phosphorolysis of GlcNAc-b1,4-GlcNAc (chitobiose) to 2-acetamido-2-deoxy-a-D-glucose 1-phosphate (a-GlcNAc-1-P) and 2-acetamido-2-deoxy-D-glucose (GlcNAc). The structure-based sequence alignment with Cgs C-terminal region and V. proteolyticus ChBP generated by Phyre showed that the four regions comprising the structure of ChBP, the catalytic residue, the "fixer" residue, and residues involved in the recognition of the nucleophilic substrate (phosphate) are conserved in Cgs (SI Fig. 9). Cgs residues Thr-2775 and Ser-2777 are analogous to V. proteolyticus ChBP Thr-709 and Thr-711, respectively, and these last residues are involved in the interaction with phosphate (SI Fig. 9). This fact may explain why even the protein truncated at position 2773 with only the 94 C-terminal amino acid residues deleted shows the phenotype of CbG with high DP.

SI Materials and Methods

Bacterial Strains and Growth Conditions. Escherichia coli K-12 DH5a-F'IQ (Invitrogen) and XL1-Blue MRF' (Stratagene) strains were used as host for all plasmids used in this study. E. coli BL21-CodonPlus(DE3)-RP strain (Stratagene) was used as host for protein expression. A. tumefaciens A1045 mutant strain (with a Tn5 insertion in the chvB gene coding for cyclic b-1,2-glucan synthase) has been described (4-6).

E. coli and A. tumefaciens strains were grown in Luria-Bertani (LB) broth (7) at 37°C and 28°C, respectively. If necessary, media were supplemented with appropriate antibiotics as follows: ampicillin, 100 mg/ml for E. coli; carbenicillin, 100 mg/ml for A. tumefaciens; kanamycin, 50 mg/ml for E. coli and A. tumefaciens.

Linker Scanning Mutagenesis. An 11.3-kb XhoI-BamHI DNA fragment from pBA19 (8), containing the cgs gene and its own promoter, was ligated to pBBR1MCS-4 (9) digested with XhoI and BamHI, and the resulting plasmid was named pBA23. To eliminate an 800-pb DNA fragment from the upstream region of the cgs promoter, plasmid pBA23 was digested with XhoI and StuI, treated with T4 DNA polymerase (New England Biolabs), and religated, yielding pBA24.

Random 15-pb insertions were generated by using the linker scanning mutagenesis GPS-LS kit (New England Biolabs) according to the manufacturer's instructions (10). The mutagenesis is accomplished by introducing a transposon, the majority of which is then removed by restriction digest. Religation results in a 15-pb insertion, which includes a unique PmeI site. Briefly, four transposon mutagenesis reactions were performed in vitro with pBA24 (target DNA) and plasmid pGPS4 (transposon donor plasmid). E. coli XL1- Blue MRF' cells were transformed by electroporation and selected with chloramphenicol and ampicillin. The resulting colonies were resuspended in LB, and total plasmidic DNA was extracted from the pool of colonies (without prior overnight growth in liquid culture to prevent competition during growth and possible distortion of library representation), yielding mutant library I. All plasmids in it have the full-length transposon inserted. DNA from library I was digested with PmeI, and the digested DNA corresponding to the target plasmid without the bulk of the transposon was religated. XL1- Blue MRF' cells were transformed by electroporation and selected with ampicillin. The resulting colonies were resuspended in LB, and total plasmidic DNA was purified directly from the pool of colonies, yielding mutant library II. All plasmids in it have an insertion of 15 pb that includes the unique PmeI site. In two of three frames, this leads to an insertion of 5 aa. In this case, the resulting plasmids were named with a number indicating the position of the last residue of Cgs before the insertion. In the remaining frame, the TAA sequence of PmeI site is read as a stop codon, resulting in a truncated protein at the site of insertion; the resulting plasmids were named with a number indicating the position of the last residue of Cgs, and the word "stop." Finally, plasmids of library II were introduced into the A. tumefaciens A1045 cgs mutant strain by electroporation, and transconjugants were selected with kanamycin and carbenicillin. The position of the 15-pb insertion was determined by restriction mapping using the PmeI site generated at the insertion site and DNA sequencing.

TLC Analysis of Cyclic Glucans. For assaying in vivo activity, cells from 3 ml of stationary phase cultures of A. tumefaciens strains grown for 24 h at 28°C (200 rpm) were harvested by centrifugation. CbG were extracted from cell pellets with ethanol (70% ethanol, 1 h at 37°C). Ethanolic extracts were centrifuged, and supernatants were dried in a speed-vac centrifuge. Extracted glucans were dissolved in 70% ethanol and submitted to TLC on silica gel-60 plate (Merck) with 1-butanol/ethanol/water (5:5:4, vol/vol) as described previously (8). Sugars were detected by baking for 10 min at 125°C after dipping the plate in 5% sulfuric acid in ethanol.

Isolation and Purification of Cyclic b-1,2-Glucans. Cells from 100 ml of stationary phase cultures grown for 24 h at 28°C (200 rpm) were harvested by centrifugation at 8,000 ´ g for 10 min at 4°C. Cell pellets were extracted with ethanol (70% ethanol, 1 h at 37°C). The ethanolic extracts were centrifuged, and the supernatants were concentrated in a speed-vac centrifuge and subjected to gel filtration on a Bio-Gel P4 column (78 by 1.8 cm; Bio-Rad Laboratories). Columns were eluted at room temperature with 0.5% formic acid at a flow rate of 9 ml/h, and 1.5 ml fractions were collected. Fractions corresponding to CbG were pooled, concentrated, and applied to DEAE-Sephadex A-25 column (7 by 1 cm; Amersham Pharmacia Biosciences). Columns were eluted with 16 ml of water and 16 ml of 250 mM NaCl at a flow rate of 0.5 ml/min, and 4-ml fractions were collected. Carbohydrates were detected by the anthrone-sulfuric acid method (11).

Chromatographic Analysis. Samples were analyzed by HPAEC-PAD on a Carbopack PA-100 column equipped with a PA-100 precolumn. A ICS-3000 ion Chromatography system (Dionex Corporation) equipped with an electrochemical detector was used. Columns were eluted at a flow rate of 1 ml/min by a two-step procedure consisting of (i) 120 mM sodium acetate in 100 mM NaOH for 5 min and (ii) a linear gradient of 120 mM to 300 mM sodium acetate in 100 mM NaOH for 45 min.

MALDI-TOF Mass Spectrometry Analysis. All measurements were performed by using an Ultraflex II TOF/TOF mass spectrometer equipped with a high-performance solid-state laser (l = 355 nm) and a reflector (Bruker Daltonics, Bremen, Germany). Samples were measured in the linear and the reflectron modes and as routine in both the positive and the negative ion modes. Spectra were recorded between 0 and 5 kDa. A hundred shots were averaged for each spectrum. Calibration was performed with b-cyclodextrin (cycloheptaamylose, MW 1,135.0) and g-cyclodextrin (cyclooctaamylose, MW 1,297.1) and a peptide Calibration standard from Bruker [angiotensin II, angiotensin I, substance P, bombesin, ACTH (1-17), ACTH (18-39), and somatostatin]. Matrix solution was prepared by dissolving 1 mg of 2,5-dihydroxybenzoic acid with 0.1 ml of CH3CN/H2O (3:2, vol/vol).

Site-Directed Mutagenesis. Plasmid pBBR1MCS-4 (9) was digested with SphI, treated with T4 DNA polymerase, and religated, yielding pBBSP. An 11.3-kb XhoI-BamHI fragment from pBA19 (8), containing the cgs gene and its own promoter, was ligated in pBBSP digested with XhoI and BamHI; the resulting plasmid was named pBA25. Plasmid pBA25 was digested with SphI and SacI, and the excised 1.6-kb SphI-SacI cgs fragment was ligated to pGemT-easy (Promega) digested with the same enzymes, yielding pGTSS.

Amino acid residues Asp-2420 and Asp-2555 were replaced by alanine by using the QuikChange Site-Directed Mutagenesis method (Stratagene). The PCRs were performed by using pGTSS as template and synthetic oligonucleotides with the appropriate nucleotide changes. The PCR mixture was digested with DpnI and used to transform E. coli DH5a-F'IQ cells. The resulting plasmids were digested with SphI and SacI, and the excised 1.6-kb SphI-SacI cgs fragments containing the desired mutations were ligated into pBA25 digested with the same enzymes, yielding pD2420A and pD2555A. Mutated DNA was sequenced to confirm PCR fidelity. Plasmids pBA25, pD2420A and pD2555A were introduced into A. tumefaciens A1045 cgs mutant strain by electroporation.

Cloning, Expression, and Purification of Recombinant Cgs C-Terminal Domain. Plasmid pTrcHisA (Invitrogen) was digested with SphI, treated with T4 DNA polymerase, and religated, yielding pTrcHisA-SphI(-). Plasmid p1493 (containing the cgs gene with a 15-pb insertion at the position corresponding to the amino acid residue Met-1493) was digested with PmeI and SacI, and the excised fragment of 4.5 kb was ligated to plasmid pTrcHisA-SphI(-), which was digested with BamHI, treated with T4 DNA polymerase, and then digested with SacI. The resulting plasmid, which codifies for the C-terminal domain of Cgs (residues 1493-2867) with an N-terminal (His)6 tag, was named pCT. Plasmids pD2420A and pD2555A were digested with SphI and SacI, and the excised fragments of 1.6 kb containing the indicated mutation were ligated to plasmid pCT digested with the same enzymes, yielding pCT-D2420A and pCT-D2555A, respectively. E. coli BL21-CodonPlus(DE3)-RP strain (Stratagene) cells were transformed with each construct (pCT, pCT-D2420A, and pCT-D2555A), and protein expression was induced with 0.2 mM IPTG during 7 h at 18°C. Recombinant soluble proteins were purified by using Ni-NTA (Invitrogen) affinity chromatography. Eluates containing the corresponding purified proteins were dialyzed against 50 mM Tris·HCl buffer (pH 7.6)/250 mM NaCl.

Phosphorylase Enzyme Assay. Phosphorylase activity was determined by measuring the amount of glucose 1-P formed by phosphorolysis of the substrate by a coupled enzyme assay measuring the formation of NADPH at 340 nm. The reaction mixture contained 50 mM Tris·HCl buffer (pH 7.6), 5 mM MgCl2, 30 mM phosphate buffer (pH 7.6), 3 mM NADP, 30 mM DTT, 5 mM glucose 1,6-bisphosphate (Sigma), 4 units·ml-1 phosphoglucomutase (Sigma), 2 units·ml-1 glucose 6-phosphate dehydrogenase (Sigma), and the indicated amounts of substrate and recombinant enzyme in a total volume of 0.1 ml. Formation of NADPH was followed continuously with a spectrophotometer during 5 min at 25°C. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 mmol of glucose 1-P per min. Partial acid-hydrolyzed CbG were used as substrate for the reaction. Partial acid hydrolysis of CbG was carried out in 0.5 N HCl at 100°C for 5 min. All proteins used in this assay were purified at the same time, following the same protocol.

Isolation and Characterization of Glycopeptides. Total membrane fractions of A. tumefaciens A1045 cgs mutant strain harboring plasmids pBA25, pD2420A, or pD2555A were incubated with UDP-[14C]glucose (500,000 cpm; 300 mCi/mmol) in 50 mM Tris·HCl buffer (pH 8.2)/5 mM MgCl2 at 28°C for 20 min, as described previously (12). The reaction was stopped with 10% trichloroacetic acid (TCA). Washed TCA precipitates were treated at 37°C with 2 mg of type XIV protease from Streptomyces griseus (Sigma) in 100 mM Tris·HCl buffer (pH 7.6)/10 mM CaCl2 in a total volume of 1 ml. After 72 h of incubation, 100 ml of 100% TCA was added to stop the reaction, and [14C]glucose-labeled glycopeptides were recovered from the supernatant and subjected to gel filtration on a Bio-Gel P4 column (78 by 1.8 cm). The column was eluted with 0.1 M pyridine-acetate buffer (pH 5.5) at a flow rate of 9 ml/h, and 1.5-ml fractions were collected. Radioactivity was quantified by liquid scintillation.

Incubation of Glycopeptides with the Recombinant C-Terminal Domain of Cgs. [14C]Glucose-labeled glycopeptides (3,400 cpm) recovered from the Bio-Gel P4 column (see Isolation and Characterization of Glycopeptides) were incubated with the recombinant C-terminal domain (Cgs-CT, 10 mg) for 1 h at 25°C in 50 mM Tris·HCl buffer (pH 7.6), 30 mM phosphate buffer (pH 7.6), and 30 mM DTT in a total volume of 0.1 ml. After heating the mixture for 1 min at 100°C, 300 ml of deionized water was added, and the radioactive products were analyzed by chromatography on a DEAE-Sephadex A-25 column (3 by 0.6 cm; Amersham Pharmacia Biosciences). The column was eluted with 1 ml of water and with 1.0 ml each of 50, 100, 150, 200, 250, and 500 mM NaCl. Fractions of 1.0 ml were collected, and radioactivity was quantified by liquid scintillation.

Analysis of the radioactive products by HPAEC was performed on a Carbopack PA-10 column equipped with a PA-10 precolumn. An isocratic elution consisting of 100 mM NaOH and 12 mM sodium acetate was performed at a flow rate of 1 ml/min. Fractions of 0.3 ml were collected, and radioactivity was quantified by liquid scintillation.

Incubation of Total Membrane Fractions with [32P]-inorganic Phosphate. Total membrane fractions of A. tumefaciens A1045 cgs mutant strain harboring plasmids pBA24, p1587stop, or pD2420A were incubated with NaH232PO4 (222,000 cpm; 1 mCi/mmol) in 50 mM Tris·HCl buffer (pH 8.2), 5 mM MgCl2, and 5 mM UDP-glucose for 1 h at 28°C in a total volume of 0.05 ml. To stop the reaction 100 ml of 100% ethanol was added, the samples were centrifuged, and the radioactive products in the supernatant were analyzed by paper electrophoresis with 1.2 M pyridine acetate buffer (pH 6.5) during 1.5 h at 1,000 V. Radioactivity was detected by autoradiography. Sugars were detected by the alkaline-silver method (13). Compounds containing phosphorus were detected by Burrows reagent (14). UDP-glucose and UTP standards were detected by UV. Mild acid hydrolysis was carried out in 0.1 N HCl at 100°C for 10 min.

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This Article

  1. Published online before print October 5, 2007, doi: 10.1073/pnas.0708025104 PNAS October 16, 2007 vol. 104 no. 42 16492-16497
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