Biosci. Biotechnol. Biochem., 75 (1), 171–174, 2011
Note
Binding Specificity of the Recombinant Cytoplasmic Domain of Cordyceps militaris -1,3-Glucan Synthase Catalytic Subunit Minoru UJITA,y Ryosuke INOUE, Yusuke M AKINO, Yosuke K ATSUNO, and Hiroki O KUMURA Laboratory of Biological Chemistry, Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan Received September 14, 2010; Accepted October 12, 2010; Online Publication, January 7, 2011 [doi:10.1271/bbb.100660]
The cytoplasmic domain of the medicinal mushroom Cordyceps militaris -1,3-glucan synthase catalytic subunit Fks1 was expressed as a fusion protein with an Nterminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and was assayed for binding specificity. The recombinant cytoplasmic domain bound specifically to UDP-agarose and lichenan ( -glucan), but not to ADP-agarose, GDPagarose, or other carbohydrates. Key words:
Cordyceps militaris; -1,3-glucan synthase; catalytic subunit; cytoplasmic domain; binding specificity
The cell wall -glucans of yeast, filamentous fungi, and mushrooms are essential for their shape and viability, and some mushroom -glucans stimulate immune responses and potentially have tumor-inhibitory effects in humans.1) Lentinan, PSK (krestin), and schizophyllan are beneficial in clinics used in conjunction with chemotherapy. The entomopathogenic fungus Cordyceps militaris is found in vegetable wasps and plant worms and is used as a tonic food and herbal medicine, but the molecular mechanisms of its pharmacological activities are not fully understood. The major structural components of the fungal cell wall are -1,3and -1,6-glucans, chitin, mannan, and glycoproteins having high mannose-type glycans, and among these 1,3-glucan is the most prevalent. -1,3-Glucan is synthesized from uridine 50 -diphosphate (UDP)-glucose by a membrane protein complex, -1,3-glucan synthase, which is stimulated by GTP. It appears that -1,3-glucan biosynthesis occurs on the cytoplasmic side of the plasma membrane and that -1,3-glucan chains are extruded toward the periplasmic space. The -1,3glucan synthase complex has been found to be composed of catalytic subunit Fks, a large-molecular-size polypeptide with transmembrane domains, and regulatory subunit Rho1, a small-molecular-size GTP-binding protein (GTPase), that stimulates -1,3-glucan synthase activity in its prenylated form.2–5) Recently we isolated cDNA clones encoding C. militaris -1,3-glucan synthase catalytic subunit Fks1 (CmFks1), an integral membrane protein, and Rho1 (CmRho1).6,7) CmFks1 is a 1981-amino acid protein that shows significant
similarity to other fungal Fks proteins. Similarly to Saccharomyces cerevisiae Fks1, CmFks1 possesses 16 potential transmembrane domains with a relatively large hydrophilic domain in the middle of the protein. The region of CmFks1 most homologous to S. cerevisiae Fks1 is a large hydrophilic domain of 578 amino acids that is predicted to be a cytoplasmic domain. This domain is a candidate for the location of the catalytic site. As reported for other fungal Fks proteins, CmFks1 does not contain the proposed UDP-glucose binding motif QXXRW, but it has regions with homology to BcsAp, the catalytic subunit of cellulose synthase from Acetobacter xylinium.8) These specific domains (domains 1 and 2) are located in the hydrophilic central region of CmFks1.6) Because cellulose synthase catalyzes the formation of -1,4-glucan using UDP-glucose as substrate, these conserved domains may be involved in UDP-glucose binding, but it is not known whether the cytoplasmic domain of fungal Fks1 binds to substrates such as UDP-glucose and -1,3-glucan. Because cellwall -1,3-glucan is specific and essential to fungal life, -1,3-glucan synthase is a possible target for the design of novel chemotherapeutics against human pathogens such as fungi. -1,3-Glucan synthase inhibitors might affect essential process in fungal life functions. Hence in vitro assay systems must be established to characterize the binding specificity of the catalytic site of fungal 1,3-glucan synthase. Such assay systems can also be used to screen -1,3-glucan synthase inhibitors as fungicides. Here we report the substrate binding specificity of the cytoplasmic domain of CmFks1. This is the first report that the cytoplasmic domain of fungal -1,3glucan synthase catalytic subunit Fks1 binds to UDP and the -glucan such as lichenan, a mixed-linkage -glucan with laminarioligosaccharide structures. To produce a hexahistidine (His6 )-tagged glutathione S-transferase (GST)–CmFks1 fusion protein, an E. coli cell-free translation system expression plasmid encoding the cytoplasmic domain of CmFks1 was constructed. The cDNA encoding the C. militaris Fks1 cytoplasmic domain (CmFks1CD) was prepared by polymerase chain reaction (PCR) using C. militaris cDNA as a template, based on the published nucleotide sequence.6) The sense and antisense primers for this PCR were 50 -GGCCATGGGGCGAAATATCTTTTCGAGACTGC
y To whom correspondence should be addressed. Fax: +81-52-833-5524; E-mail:
[email protected] Abbreviations: BSA, bovine serum albumin; CBB, Coomassie Brilliant Blue; CmFks1, Cordyceps militaris -1,3-glucan synthase catalytic subunit Fks1; CmFks1CD, Cordyceps militaris Fks1 cytoplasmic domain; Fuc, L-fucose; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; GST, glutathione S-transferase; His6 , hexahistidine; HRP, horseradish peroxidase; Man, D-mannose; PCR, polymerase chain reaction; TBS, Tris-buffered saline
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and 5 -GGCCCGGGTCAGTTGTTGACGTGGAAGCCCGCGTG (NcoI and SmaI sites underlined). The PCR product encoding amino acid residues 781–1358 of CmFks1 was digested with NcoI and SmaI, and then cloned in the NcoI and SmaI sites of pIVEXGST (Roche), yielding pIVEX-GST/CmFks1CD. Recombinant pIVEX-GST/CmFks1CD was introduced into competent E. coli JM109 cells. The nucleotide sequence of the expression construct was analyzed by dideoxy nucleotide sequencing to ensure absence of errors. The His6 -tagged GST-CmFks1 fusion protein (HisGST-CmFks1CD) or His6 -tagged GST alone (His-GST) was expressed in an E. coli cell-free translation system (RTS E. coli Disulfide Kit, Roche) and purified using Glutathione Sepharose 4B (Microspin GST Purification Module, GE Healthcare). The purified fusion protein was analyzed by 10% SDS–PAGE and immunoblotting. The proteins in the gel were stained with Coomassie Brilliant Blue (CBB) or transferred to a nitrocellulose membrane. The membrane was then stained with horseradish peroxidase (HRP)-conjugated anti-GST antibody (GE Healthcare) to detect GST fusion proteins. Positive bands were visualized using the enhanced chemiluminescence (ECL) detection system (GE Healthcare). The His-GST-CmFks1CD fusion protein was purified and used to test for binding activity. SDS–PAGE and immunoblot analysis showed that the purified fusion protein had the expected molecular size of 93 kDa and was detectable with anti-GST antibody (Fig. 1). To determine the substrate binding activity of HisGST-CmFks1CD, containing the cytoplasmic domain of the C. militaris -1,3-glucan synthase catalytic subunit, UDP-agarose (Glycosyltransferase Affinity Gel-UDP, Calbiochem) was washed and suspended with Tris-buffered saline (TBS) (150 mM NaCl, 10 mM Tris–HCl, pH 7.5) containing 0.05% (v/v) Triton X100, 1% bovine serum albumin (BSA), and 10 mM CaCl2 . Purified His-GST-CmFks1CD was added to the UDP-agarose suspension, and the mixture was incubated for 30 min at room temperature with continuous end-over-end mixing.9) Following incubation, the insoluble UDP-agarose was pelleted by centrifugation, and the supernatants were assayed for GST activity. GST activity was assayed using a GST detection module (GE Healthcare). The purified His-GSTCmFks1CD was also incubated without the UDPagarose suspension and assayed for GST activity. As a control, the binding activity of His-GST alone was tested under the same conditions. Each assay was done in triplicate. The binding specificity of CmFks1CD was determined by incubating the purified His-GST-CmFks1CD with the following insoluble polysaccharides and gels: ADPagarose (Sigma), GDP-agarose (Glycosyltransferase Affinity Gel-GDP, Calbiochem), chitin beads (New England Biolabs), lichenan, curdlan, cellulose, mannanagarose, heparin-agarose, deaminated heparin-agarose, xylan, -glucose-agarose, N-acetylglucosamine (GlcNAc)agarose, mannose-agarose, N-acetylgalactosamine (GalNAc)-agarose (Sigma), chitooligo-agarose, lactose-agarose, melibiose-agarose, and fucose-agarose (Seikagaku, Tokyo). The binding assay was done as described above.
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Fig. 1. Expression and Purification of His-GST-CmFks1CD Fusion Protein. A, Model of the predicted membrane topology of CmFks1 (top) and schematic representation of structural domains of His-GSTCmFks1CD fusion protein (bottom). The plasma membrane is shown as a long rectangle filled with wavy lines, and the solid black line represents the polypeptide chain with its putative outer and inner loops.6) The 16 predicted transmembrane helices are shown as vertical black bars.6) His and GST represent hexahistidine and glutathione S-transferase respectively. B, The glutathione-Sepharose-purified His-GST-CmFks1CD fusion protein was electrophoresed on a 10% SDS-polyacrylamide gel and then stained with CBB (lane 1) or transferred to a nitrocellulose membrane. The membrane was stained with anti-GST antibody (lane 2). Molecular mass markers are shown to the left.
In some cases, competing carbohydrates (50 or 100 mM monosaccharides, 50 or 100 mM oligosaccharides, and 0.5 or 1 mg/mL polysaccharides) were added to the reactions. First we examined the ability of soluble His-GSTCmFks1CD to recognize UDP-agarose. His-GSTCmFks1CD bound to UDP-agarose, but did not bind to ADP-agarose or GDP-agarose (Fig. 2A). These data indicate that His-GST-CmFks1CD specifically recognizes UDP, but not ADP or GDP. Furthermore, HisGST-CmFks1CD bound to lichenan, but did not bind to curdlan, cellulose, chitin, mannan-agarose, heparinagarose, deaminated heparin-agarose, xylan, chitooligoagarose, lactose-agarose, melibiose-agarose, -glucoseagarose, GlcNAc-agarose, mannose-agarose, GalNAcagarose, or fucose-agarose (Fig. 2B). This indicates that His-GST-CmFks1CD specifically recognizes lichenan among the carbohydrates tested. His-GST, the other portion of the fusion protein, did not bind to UDP-agarose or lichenan (Fig. 2), indicating that the observed binding activity of the fusion protein was due to the portion corresponding to CmFks1CD. The binding of His-GST-CmFks1CD to lichenan was dose-dependently inhibited by the addition of glucose, but not by the other monosaccharides tested in this study (Fig. 3A), indicating that CmFks1CD specifically recognizes glucose residues in the -glucan, such as lichenan, a mixed-linkage -glucan with laminarioligosaccharide structures. In addition, -glucan binding
Functional Expression of Fks1 Cytoplasmic Domain
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Fig. 2. Interactions of Recombinant Soluble CmFks1CD with UDP and -Glucan. Purified His-GST-CmFks1CD or His-GST alone was incubated with the indicated insoluble gels and centrifuged, and then the supernatant was assayed for GST activity. The GST activity remaining in the sample solution after incubation with and subsequent removal of insoluble gels was expressed as percentage of total activity in the same sample solution without precipitation with insoluble gels. Values are means for three independent experiments.
of His-GST-CmFks1CD was dose-dependently inhibited by laminaribiose (with -1,3-glucosidic linkages), but not by laminarin (a soluble -1,3-glucan with -1,6-branches), glycogen (a soluble -1,4-glucan with -1,6-branches), cellobiose (with -1,4-glucosidic linkages), maltose (with -1,4-glucosidic linkages), or isomaltose (with -1,6-glucosidic linkages) (Fig. 3B), suggesting that CmFks1CD specifically recognizes the oligosaccharide structure with -1,3-glucosidic linkages in lichenan. His-GST-CmFks1CD bound to lichenan ( -1,3- and -1,4-glucan), but not to curdlan ( -1,3-glucan) or cellulose ( -1,4-glucan) (Fig. 2B), suggesting that CmFks1CD recognizes the laminarioligosaccharide structure, but does not bind -1,3-glucan, probably due to the ability to extrude -1,3-glucan chains toward the periplasmic space. While curdlan is a linear -1,3glucan, lichenan is a mixed-linkage -glucan composed of -1,3- and -1,4-D-glucopyranose residues, and contains laminarioligosaccharide structures interrupted
Fig. 3. Carbohydrate Binding Specificity of Recombinant CmFks1CD. To determine the binding specificity of CmFks1CD, the ability of various carbohydrates to block the -glucan binding of His-GSTCmFks1CD was examined. Binding assay was done as described in the legend to Fig. 2, except for the addition of competing carbohydrates. Competing monosaccharides (A) and competing oligosaccharides (B) were present at concentrations of 50 mM (open bars) and 100 mM (black bars) in the reactions, as indicated, to inhibit the binding of His-GST-CmFks1CD to lichenan. Competing polysaccharides were present at concentrations of 0.5 mg/mL (open bars) and 1 mg/mL (black bars) in the reaction mixtures, as indicated, to inhibit lichenan binding of His-GST-CmFks1CD (B). Values are means for three independent experiments. Lam2 , laminaribiose; Cel2 , cellobiose; Mal2 , maltose; Iso2 , isomaltose.
by the -1,4-glucosidic linkage. It is thought that CmFks1CD binds to the laminarioligosaccharide moiety but not to -1,3-glucan chain as a final product. These characteristics are probably required for efficient, continuous -1,3-glucan chain biosynthesis. His-GSTCmFks1CD did not bind to the other polysaccharides, polysaccharide-agaroses, oligosaccharide-agaroses, or monosaccharide-agaroses tested in this study (Fig. 2B), indicating that the soluble CmFks1CD fusion protein can be used as a tool in elucidating the biosynthetic pathway of C. militaris -glucans due to its strict ligand specificity. This recombinant CmFks1CD can be utilized to examine substrate specificity, kinetics, and reaction profile of C. militaris -1,3-glucan synthase. The protein can also be used to investigate the interactions with Rho1. Further studies, including sitedirected mutagenesis of CmFks1CD, are required to determine the substrate-binding site. The ability of HisGST-CmFks1CD to bind specifically to UDP and lichenan strongly suggests that the catalytic site of fungal -1,3-glucan synthase is located in Fks1 cytoplasmic domain.
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Acknowledgments
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This work was supported in part by the Fund for Agriomics Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Dr. Akira Hara and Dr. Eiji Yokoyama for useful discussion.
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