Glycobiology Advance Access originally published online on June 17, 2008
Glycobiology 2008 18(9):727-734; doi:10.1093/glycob/cwn053
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Characterization of two different endo-
-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens
2 Graduate School of Biostudies, Kyoto University, Kyoto 606-8502
3 The Noguchi Institute, Tokyo 173-0003
4 Faculty of Applied Biological Science, Gifu University, Gifu 501-1193, Japan
1 To whom correspondence should be addressed: Tel: +81-75-753-4298; Fax: +81-75-753-9228; e-mail: ashida{at}lif.kyoto-u.ac.jp
Received on April 25, 2008; revised on May 27, 2008; accepted on June 2, 2008
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Abstract |
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Endo-
-N-acetylgalactosaminidase (endo--GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between -GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo--GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Galβ1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo--N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415–37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl--glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAcβ1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAc, core 8 disaccharide Gal1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.
Key words:
endo--N-acetylgalactosaminidase
/
endoglycosidase
/
GH101
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mucin
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O-glycan
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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The epithelial cells of the gastrointestinal tract secrete mucous glycoproteins, which function as a barrier to pathogens, digestive enzymes, acids, and other environmental factors. The majority of oligosaccharides on mucous glycoproteins are mucin-type O-glycans, in which
-GalNAc binds the hydroxyl group of Ser or Thr residues of polypeptides. In most cases, GalNAc linked to Ser/Thr is modified by the addition of sugars such as Gal, GalNAc, and GlcNAc to form eight different types of core structures (Van den Steen et al. 2000
). They can be further elongated to more complex oligosaccharides by the attachment of these sugars (Gal, GalNAc, and GlcNAc) and terminated by the addition of Fuc, sialic acid, or sulfate. Endo--N-acetylgalactosaminidase (endo--GalNAc-ase; EC 3.2.1.9
[EC]
7) catalyzes the hydrolysis of the O-glycosidic bond in mucin-type O-glycan between -GalNAc and Ser/Thr to release oligosaccharides. To date, the enzyme activity has been reported in several bacteria: i.e., Diplococcus pneumoniae (Bhavanandan et al. 1976), Alcaligenes sp. (Fan et al. 1988), Bacillus sp. (Ashida et al. 2000), Streptomyces sp. (Ishii-Karakasa et al. 1992), and so on. Recently, we found that the enzyme is widely distributed in the enterobacterial genus Bifidobacterium, and subsequently cloned the gene engBF encoding endo--GalNAc-ase from Bifidobacterium longum JCM1217 (Fujita et al. 2005). Based on the findings of EngBF, a new glycoside hydrolase (GH) family 101 was established in the CAZy database (http://www.cazy.org/). EngBF is predicted to be an extracellular membrane-bound enzyme, which specifically acts on core 1-type O-glycan to release the disaccharide Galβ1-3GalNAc. The released disaccharide may be transported into the cytosol of bacterial cells through an ABC-type transporter specific for the disaccharides Galβ1-3GalNAc/Galβ1-3GlcNAc, which has been characterized by us (Wada et al. 2007; Suzuki et al. 2008) and metabolized by Galβ1-3GalNAc/Galβ1-3GlcNAc phosphorylase (Kitaoka et al. 2005) and N-acetylhexosamine 1-kinase (Nishimoto and Kitaoka 2007) in the cytosol. Thus, EngBF may play an important role in the degradation and utilization of mucins having core 1 O-glycans. Following a database search using the amino acid sequence of EngBF, we found a similar gene in the genome of Clostridium perfringens, a pathogenic enterobacterium. This organism is also an anaerobic bacterium found in the intestinal tract of humans and animals and causes not only diarrhea but also gas gangrene, and is thus referred to as a flesh eater. We therefore speculated that this pathogenic bacterium may have different characteristics to that of bifidobacteria regarding mucin degradation. Here, we describe the molecular cloning and characterization of a novel endo--GalNAc-ase from C. perfringens, which shows rather wide substrate specificity compared to the orthologous enzyme from bifidobacteria. |
Results |
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Molecular cloning of CPE0693 from C. perfringens
In the GH family 101, we focused on the gene CPE0693 from C. perfringens strain 13 (Shimizu et al. 2002
), which shows 29% identity in an amino acid level to EngBF from B. longum JCM1217. The open reading frame consisting of 5058 bp encodes a 1686-amino-acid protein, which is predicted to contain an N-terminal signal sequence (amino acids 1–29), Ig-like domain (amino acids 234–292), GH101 conserved region (amino acids 712–1430), and fibronectin-type III (FN3) domain (amino acids 1618–1684). In the GH101 conserved region, a carbohydrate binding module (CBM) family 4_9 domain (amino acids 1161–1313) was designated by the BLAST program (Figure 1A). This protein was predicted to be a soluble protein without any transmembrane region by the SOSUI and TMpred programs. Analyses of the flanking region of the CPE0693 gene revealed that there is a putative Shine-Dalgarno sequence (AGGGGGGAG) at –13 position from the first A of the initiation codon and this gene may not form an operon with other genes.
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We cloned the gene CPE0693 by high-fidelity PCR using the genomic DNA from C. perfringens strain 13 as a template. The recombinant protein without predicted N-terminal signal peptide (amino acids 1–29) was expressed as an N-terminally MBP (maltose binding protein)-tagged protein in Escherichia coli and affinity purified by amylose resin column chromatography. The MBP tag at the N-terminus was removed by Factor Xa protease digestion and further purified by gel filtration. The purified protein migrated as a single protein band around 185 kDa on the reducing SDS–PAGE, which is consistent with the calculated molecular mass (Figure 1B). Since the recombinant enzyme released Galβ1-3GalNAc from Galβ1-3GalNAc
1-pNP (Core 1-pNP), as revealed by thin layer chromatography (TLC) analysis (Figure 1C), we designated the gene as engCP (GenBank/EMBL/DDBJ accession number AB427163
[GenBank]
).
Characterization of EngCP from C. perfringens
To analyze the substrate specificity of EngCP in detail, we chemically synthesized a series of p-nitrophenyl (pNP) substrates with all eight types of core structures of mucin O-glycans as well as nonnatural substrates using the di-tert-butylsilylene-directed method (Imamura et al. 2005, 2006), which allows the selective synthesis of -linked GalNAc glycoside. The hydrolyses were monitored by the colorimetric measurement of released pNP and also by TLC (Table I and Figure 2). EngCP preferably hydrolyzed the core 1 substrate Galβ1-3GalNAc1-pNP, similar to EngBF. Interestingly, however, the enzyme hydrolyzed the core 2 trisaccharide substrate Galβ1-3(GlcNAcβ1-6)GalNAc1-pNP, which was completely resistant to EngBF. To confirm the release of the intact trisaccharide, we purified the released sugars using normal-phase high-performance liquid chromatography (HPLC) (Figure 3A, bottom chart) and analyzed these compounds using MALDI-TOF/MS (Figure 3B). The molecular ion peaks appeared at m/z 609.2177 for (M + Na)+ and 625.1933 for (M + K)+, indicating that the trisaccharide was directly released from the substrate. Furthermore, EngCP released the core 3 disaccharide GlcNAcβ1-3GalNAc and the monosaccharide GalNAc. The core 8 disaccharide Gal1-3GalNAc was released very slowly. The enzyme also hydrolyzed nonnatural substrates Glcβ1-3GalNAc-pNP and GalNAcβ1-3GalNAc-pNP, and the former was the best among all the pNP substrates tested.
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The optimum pH of the enzyme for Galβ1-3GalNAc
1-pNP hydrolysis was observed at pH 6.0 and 9.0, and more than 80% activity was shown between these two peaks. In contrast, the optimum pH for the other substrates hydrolyzed was around pH 6.0. The enzyme showed highest activity at 60°C and was stable below 37°C. The Km and kcat values for Galβ1-3GalNAc1-pNP were 51 µM and 110 s–1, respectively. EngCP was severely inhibited by 1.0 mM Cu2+ but not by the same concentration of the other divalent cations tested, Ca2+, Zn2+, Co2+, Mn2+, Mg2+, Cu2+, and Fe2+, or the chelating reagent EDTA.
Comparison of substrate specificities and other characteristics between EngBF and EngCP
Previously, we reported that EngBF from B. longum showed strict substrate specificity for the core 1 O-glycan (Fujita et al. 2005). Here we re-evaluated the specificity of EngBF using newly synthesized pNP substrates (Table I). EngBF was found to act slowly on the core 3 substrate although the rate was only 0.3% of that for the core 1 substrate. The enzyme also released nonnatural Glcβ1-3GalNAc and GalNAcβ1-3GalNAc, but never acted on core 2, core 8, and -GalNAc, which were released by EngCP. The optimum temperature and thermal stability of EngBF were almost the same as that for EngCP. Optimal pH for all hydrolyzable substrates was pH 5.0–6.0. EngBF was not significantly inhibited by the divalent cations tested.
Action of EngCP on native glycoproteins
We examined the action of EngCP on intact glycoproteins. First, we incubated the enzyme with fetuin, which is known to have disialylated core 1 O-glycans (Ashida et al. 2000). When fetuin was incubated with EngCP, no released oligosaccharide was detected on TLC, suggesting that this enzyme does not act on sialyl core 1 O-glycans (Figure 4A, lane 9). We then incubated asialofetuin with EngCP, which resulted in the release of the disaccharide (Figure 4A, lane 6). EngBF also released the disaccharide from asialofetuin, but not from fetuin (Figure 4A, lanes 5 and 8). Next, we tested whether EngCP acts on porcine gastric mucin, which is known to have core 1 and core 2 O-glycans with complex modifications of sialic acid and neutral sugars. In particular, O-glycans of gastric mucin contain unique -linked GlcNAc at their nonreducing ends (Ashida et al. 2001). Although EngCP did not release oligosaccharides from intact gastric mucin, we detected the release of the core 1 disaccharide from the gastric mucin pretreated with commercial sialidase and with the recombinant exo--N-acetylglucosamindase cloned from the same strain of C. perfringens (Fujita et al. 2007) (Figure 4B, lanes 6 and 9, respectively). This result indicated that EngCP is able to remove O-glycans from gastric mucin by cooperative actions of -N-acetylglucosamindase and sialidase. However, we did not detect the release of core 2 trisaccharide from the mucin using TLC analysis. EngBF also released the disaccharide from the mucin treated in a similar way to that of EngCP (Figure 4B, lanes 5 and 8). We have detected sialidase activity in several bifidobacterial strains (unpublished data), but have never detected -N-acetylglucosamindase activity. Thus, C. perfringens, compared with bifidobacteria, might be able to release larger amounts of disaccharide from gastric mucin due to the activities of sialidase and -N-acetylglucosamindase.
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Action of EngCP on the core 1 O-glycan on cell surface glycoproteins
To examine whether EngCP could remove the core 1 disaccharide from cell surface glycoproteins, we employed Chinese hamster ovary (CHO) cells, which typically carry mono- and disialylated core 1 O-glycans (Bäckström et al. 2003
), and analyzed them by fluorescent-activated cell sorter (FACS). CHO 3B2A cells stably expressing two human glycosylphosphatidylinositol (GPI)-anchored proteins, decay accelerating factor (DAF) and CD59, were treated with either Arthrobacter sialidase or EngCP, or both, and then stained with FITC-conjugated PNA lectin. Since DAF contains a Ser/Thr-rich domain with many O-glycans at the C-terminal stalk region (Lukacik et al. 2004), CHO 3B2A cells express extra O-glycans in addition to their endogenous O-glycans. Untreated cells were not stained with PNA, suggesting that PNA-epitopes might be masked with other sugars (Figure 5A and B, dash lines). In contrast, after sialidase treatment, the fluorescent intensity was greater, indicating the exposure of PNA-epitopes by removing sialic acid at the nonreducing termini of glycans (Figure 5A and B, thin lines). When cells were treated with both sialidase and EngCP, the fluorescent intensity decreased but was still higher than that of untreated cells (Figure 5A, thick line). Since PNA lectin binds both Galβ1-3GalNAc1- and Galβ1-3GlcNAcβ1-, and the latter epitope is commonly found in the nonreducing termini of asialo-complex-type N-glycans, the remaining fluorescent intensity in the case of the two enzyme-treated cells might be derived from N-glycans, which are not hydrolyzed by EngCP. EngBF showed similar results to EngCP (Figure 5B, thick line).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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In this paper, we have cloned and characterized EngCP, an endo-
-GalNAc-ase from C. perfringens, and compared it with the orthologous enzyme EngBF from B. longum. Both enzymes preferably released the core 1 disaccharide Galβ1-3GalNAc from mucin-type O-glycans. Detailed analyses using systematically synthesized substrates revealed that these enzymes also released the core 3 disaccharide, GlcNAcβ1-3GalNAc, and nonnatural disaccharides, Glcβ1-3GalNAc and GalNAcβ1-3GalNAc. These results indicate that both enzymes require the substitution at C3 position of (–1)GalNAc by β-hexose (β-Gal/Glc) or β-N-acetylhexosamine (β-GalNAc/GlcNAc). Exceptionally, EngCP acted on GalNAc1-pNP, suggesting that this enzyme is more likely to tolerate C3 substitution. In both enzymes, 2-acetamide substitution of (–2)Gal in the core 1 disaccharide caused a large reduction in activity, whereas a 4-epimeric change of (–2)Gal caused a small reduction in EngBF but high activation in EngCP. The co-occurrence of 2-acetamide substitution and 4-epimeric change of (–2)Gal caused a severe reduction in the activity of both enzymes; however, EngCP still showed a 10-fold higher activity than EngBF. The most striking difference was that the core 2 trisaccharide was released by EngCP but not by EngBF. In addition, EngCP released the monosaccharide -GalNAc and the core 8 disaccharide. Taken together, these results suggest that EngCP may possess a rather larger pocket in the active site and thus showed relatively broader substrate specificity compared to EngBF.
Clostridia and bifidobacteria both reside in the intestines of humans and animals. The former are largely pathogenic bacteria, whereas the latter are believed to be probiotic bacteria, which are beneficial to the host. How does endo--GalNAc-ase function in the gut? It was reported that gastric and duodenal mucins contain core 1 and core 2 O-glycans, whereas small and large intestinal mucins mainly contain core 3 O-glycan (Brockhausen 2006). Thus, the core 1 releasing activity of EngBF and EngCP may function in digestion of detaching mucins, which flow from the stomach. However, unmasked core 1 and core 2 O-glycans may not be frequently found, and instead almost all O-glycans are modified by other sugars such as Gal, GalNAc, GlcNAc, Fuc, and sialic acid. In fact, bifidobacteria show activities of β-galactosidase (Rossi et al. 2000; Moller et al. 2001), β-N-acetylhexosaminidase (unpublished data), 1,2--fucosidase (Katayama et al. 2004), and sialidase (unpublished data), indicating that the above sugars attached to the core 1 disaccharide may be released by these exoglycosidases to expose unmasked disaccharide. In contrast, C. perfringens possesses not only these exoglycosidases but also a variety of endoglycosidases, i.e., several specific endo-β-galactosidases. Sugar chains of mucin glycoproteins are frequently capped by terminal glyco-epitopes, such as ABH blood group antigens, GlcNAc1-4Galβ1-, and Gal1-3Galβ1- (pig xenoantigen). These glyco-epitopes can be removed by the following endoglycosidases from C. perfringens: blood groups A- and B-trisaccharide-releasing endo-β-galactosidase (Anderson et al. 2005), GlcNAc1-4Gal-releasing endo-β-galactosidase (Ashida et al. 2001, 2002), and Gal1-3Gal-releasing endo-β-galactosidase C (Ogawa et al. 2000), respectively. Such endo-β-galactosidase activities were not detected in bifidobacteria (unpublished data). These facts suggest that clostridia have an advantage as residents in the intestinal tract compared with other intestinal bacteria.
In the large intestine where clostridia and bifidobacteria reside, the core 3 structure may be abundant in mucin O-glycans (Robbe et al. 2004; Brockhausen 2006). EngCP released the core 3 disaccharide approximately 10-fold higher than EngBF. Thus, C. perfringens may be able to remove almost all types of gastrointestinal mucin O-glycans, whereas B. longum prefers rather shorter and limited oligosaccharides. With regard to bifidobacteria, it was recently reported that core 1 disaccharides are transported into the bacterial cells via an ABC-type transporter specific for Galβ1-3GalNAc/Galβ1-3GlcNAc (Wada et al. 2007; Suzuki et al. 2008) and are further degraded to Gal1-phosphate and GalNAc by Galβ1-3GalNAc/Galβ1-3GlcNAc specific phosphorylase in the cytosol of the cells (Kitaoka et al. 2005; Nishimoto and Kitaoka 2007). On the other hand, C. perfringens was also reported to possess a similar phosphorylase which was more highly specific for Galβ1-3GalNAc (Nakajima et al. 2008). Although the genes encoding Galβ1-3GalNAc transporter have not been found in the genome of C. perfringens, this pathogenic bacterium may be able to incorporate Galβ1-3GalNAc into the cells by an unidentified mechanism evolutionally different from bifidobacterial one.
It should also be noted that most clostridial glycosidases including EngCP are extracellular soluble enzymes, which may penetrate the mucin layer and reach the surface of epithelial cells. Thus, these enzymes could damage intestinal mucus. However, previously known bifidobacterial glycosidases are membrane or cell-wall anchored and are not liberated from bacterial cells. In conclusion, the broader substrate specificity of EngCP may contribute to the pathogenicity of C. perfringens.
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Materials and methods |
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Substrates
The pNP-
-glycosides with core 1, core 2, and core 3 structures, and GalNAc1-pNP were purchased from Toronto Research Chemicals, Canada. The other pNP-glycosides listed in Table I were chemically synthesized by the di-tert-butylsilylene-directed method (Imamura et al. 2005, 2006; Sato, Imamura, Ando, Ishida, Kiso, submitted). Fetuin and asialofetuin from fetal calf serum and mucin from porcine stomach were from Sigma-Aldrich (MO).
Cloning of CPE0693 from C. perfringens
The coding sequence for amino acid 30-1686 of CPE0693 was amplified by PCR using genomic DNA from C. perfringens strain 13 (kindly provided by Dr. T. Shimizu), a pair of primers, 5'-acgcggatccacacctgttaatgaagctg and 5'-ttttgtcgacttatcttgcagttctaacag, containing BamHI and SalI sites, respectively, and KOD-plus DNA polymerase (Toyobo, Japan). The amplified fragment was digested with BamHI and SalI and ligated into a pMAL-c2X vector (New England Biolabs, MA) to generate pMAL-c2X-engCP. The nucleotide sequence was confirmed by sequencing.
Expression and purification of the recombinant EngCP
E. coli DH5 was transformed with pMAL-c2X-engCP and cultured in a Luria-Bertani liquid medium containing 100 µg/mL ampicillin at 37°C for 12 h. The cells were harvested, suspended in a 20 mM potassium phosphate buffer (pH 7.0), and disrupted by sonic oscillation (Insonator 201M, Kubota, Japan). Cell-free extract was applied onto an Amylose Resin High Flow column (New England Biolabs). After washing with a 20 mM potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl, bound proteins were eluted with the same buffer containing 10 mM maltose. The eluted proteins were concentrated and applied onto a Superdex 200 10/300 GL column equipped on an ÄKTA explorer (GE Healthcare, England). The eluted EngCP was digested with Factor Xa protease (New England Biolabs) to remove the MBP tag and further purified by Superdex 200 10/300 GL column chromatography. The recombinant EngBF was prepared as described (Fujita et al. 2005).
Enzyme assays
Enzyme assays were carried out using a synthetic substrate Galβ1-3GalNAc1-pNP as previously described (Ashida et al. 2000; Fujita et al. 2005). For the detection of released sugars, the enzyme products were separated by silica-gel TLC (Merck 5553, Merck, Germany) with 1-butanol:acetic acid:water (2:1:1, by volume) as a developing solvent and visualized using a diphenylamine-aniline-phosphoric acid reagent (Anderson et al. 2000). For the separation of sialic acid, GlcNAc and Galβ1-3GalNAc, 1-propanol:28% ammonia:water (15:1:6, by volume) was used as a developing solvent.
HPLC
HPLC analysis was performed using a TSK-Gel Amido-80 column (4.6 x 250 mm, Tosoh, Japan). Elution was carried out with acetonitrile:water (3:1, by volume) as a solvent at a flow rate of 1.0 mL/min at 40°C, and monitored by absorbance at 214 nm.
Flow-cytometry
CHO 3B2A stably expressing human GPI-anchored proteins, CD59 and DAF, were used (Nakamura et al. 1997). Cells were cultivated in a HAM F-12 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (GIBCO, CA). Cells suspended in phosphate-buffered saline (PBS) were treated with sialidase from Arthrobacter ureafacience (Nacalai Tesque, Japan) and either EngCP or EngBF at 37°C for 30 min. After washing with PBS, the cells were stained with FITC-conjugated PNA lectin (J-Oil Mills, Japan) at 4°C for 30 min. Flow cytometry was carried out using FACS Vantage (Becton Dickinson, NJ).
MALDI-TOF/MS analysis
Mass spectra were obtained on a Bruker Daltonics Autoflex-G system (MA) with a 337-nm nitrogen laser, using the positive-ion mode, the reflectron mode, and external calibration with PEG. The sample was dissolved in ethanol at a concentration of 1 mg/mL. An -cyano-4-hydroxycinnamic acid solution (25 mg/mL acetonitrile:0.1% TFA = 3:1, by volume) was used as the matrix. Half a microliter of the mixture of these solutions (matrix:sample = 4:1, by volume) was placed on the AnchorChip target (Bruker, Germany). During the measurement, 30 laser shots were used, and the data of the mass spectra were collected at different positions of the crystallized sample spot.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency and Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Dr. T. Shimizu of Kanazawa University for providing the genomic DNA from C. perfringens strain 13, Dr. T. Kinoshita of Osaka University for CHO 3B2A cells, and Dr. S. Kurihara, Dr. K. Fujita, and Mr. A. Kawahara for discussion.
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Abbreviations |
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CHO, Chinese hamster ovary; DAF, decay accelerating factor; Endo-
-GalNAc-ase, endo--N-acetylgalactosaminidase; FACS, fluorescent-activated cell sorter; GH, glycoside hydrolase; GPI, glycosylphosphatidylinositol; HPLC, high-performance liquid chromatography; MBP, maltose binding protein; PBS, phosphate-buffered saline; pNP, p-nitrophenyl; TLC, thin layer chromatography |
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