1,3-Glucan Degradation - Applied and Environmental Microbiology

4 downloads 209646 Views 1MB Size Report
Feb 16, 2012 - ... cell wall fraction. Lam16A without a GPI anchor signal peptide was ..... clonal antibody against the polyhistidine tag (Qiagen) or the FLAG tag.
A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolase from Ustilago esculenta Functions in ␤-1,3-Glucan Degradation Masahiro Nakajima,a* Tetsuro Yamashita,b Machiko Takahashi,a Yuki Nakano,a and Takumi Takedaa Iwate Biotechnology Research Center, Iwate, Japan,a and Iwate University, Iwate, Japanb

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/ MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative ␤-1,3glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarindegrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 ␤-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward ␤-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify ␤-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of ␤-1,3-glucan after its release in the extracellular medium.



-1,3-Glucan occurs widely as a major component of the cell walls of most fungi. Extracellular ␤-1,3-glucan that forms a gel-like sheath is produced by Phanerochaete chrysosporium (35, 37, 44). Plants produce a form of cell wall-associated ␤-1,3-glucan, called callose, in response to biotic or abiotic stress (11), while some algae accumulate ␤-1,3-glucan as a storage polysaccharide (29). Several glycoside hydrolases are involved in ␤-1,3-glucan biosynthesis and degradation. The hydrolysis of ␤-1,3-glucan can be performed by the combined action of endo-␤-1,3-glucanases as well as ␤-glucosidases or exo-␤-1,3-glucanases, which are found in glycoside hydrolase family 16 (GH16), GH17, GH55, GH64, and GH81 as well as GH3, GH5, GH17, and GH55, respectively, based on the Cazy database (http://www.cazy.org/). ␤-1,3-Glucanosyltransferase from Aspergillus fumigatus, a member of GH72, catalyzes the hydrolysis of ␤-1,3-glucan and simultaneously produces an insoluble ␤-1,3-glucan from laminarioligosaccharides by a transglycosylation reaction (19). The modification of ␤-1,3-glucan by these enzymes is thought to play a significant role in cell wall morphogenesis and in nutrient (carbon) acquisition. Glycosylphosphatidylinositol (GPI) anchoring is a posttranslational lipid modification that anchors proteins to the plasma membrane. A number of examples are known, including a variety of glycoside hydrolases, such as endo-␤-1,3-glucanase and chitinase in GH16, GH17, GH72, and GH81 (13, 36, 45), and proteins involved in transmembrane signaling, e.g., receptors and adhesion molecules (3, 26, 33). The core structure of the GPI anchor moiety is highly conserved from yeast to mammals (39). GPI anchoring to proteins occurs in the endoplasmic reticulum. GPIanchored proteins then transit the secretory pathway to reach the cell surface (33). The GPI anchor moiety is further modified during transport. The remodeling process is essential for the proper association of GPI-anchored proteins with lipid microdomains (lipid rafts), areas rich in sphingolipids and sterols formed by

5682

aem.asm.org

localized phase separation in the plasma membrane (10, 40). GPIanchored proteins bound to the membrane have a longer turnover time than membrane proteins with a proteinaceous transmembrane anchor (5, 23, 38). Side chains in the GPI anchor moiety allow for a high packing density of the tethered proteins (25). Proteomic analyses have revealed that many proteins that possess a GPI anchor signal peptide are covalently linked to the cell wall (6, 12, 14, 15, 25, 48). Crh1p and Crh2p, which are putative GPI-anchored enzymes belonging to GH16, are covalently linked to the cell wall of Saccharomyces cerevisiae and catalyze the transglycosylation of chitin to ␤-1,3-glucose branches of the ␤-1,6glucan backbone (7). The deletion of genes encoding Crh family proteins caused a remarkable reduction in the amount of ␤-1,6glucan in the cell wall of Candida albicans (34). These observations suggest the involvement of Crh1p and Crh2p in cell wall organization. Thus, GPI-anchored glucoside hydrolases are found to localize to the plasma membrane or cell wall through a GPI anchor moiety. However, the significance of the hydrolytic activity of GPI-anchored glycoside hydrolases remains unknown. In this study, we identify and characterize for the first time, to our knowledge, a GPI-anchored ␤-1,3-glucanase (Lam16A) from Ustilago esculenta that is localized to the membrane via a GPI anchor but that is also secreted extracellularly. The basidiomycetous fungus Ustilago esculenta is infectious to

Applied and Environmental Microbiology

Received 16 February 2012 Accepted 25 May 2012 Published ahead of print 8 June 2012 Address correspondence to Takumi Takeda, [email protected]. * Present address: Masahiro Nakajima, Tokyo University of Science, Chiba, Japan. Supplemental material for this article may be found at http://aem.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.00483-12

p. 5682–5689

August 2012 Volume 78 Number 16

GPI-Anchored ␤-1,3-Glucanase from U. esculenta

Zizania latifolia (aquatic grass) and causes smut gall in the flowering stem, interfering with inflorescence and seed production, which results in an increase in the size and the number of host cells (9, 46). Cell wall-degrading enzymes derived from U. esculenta have been proposed to be involved in the depolymerization of cell wall polysaccharides, which induces the elongation of hyphae and results in host cell wall degradation and loosening during plant cell enlargement (8, 27, 43). The action of endo-␤-1,3-glucanase could contribute to the cell wall modification of the fungus and of the plant host as well as to saccharification in concert with ␤-glucosidases (32). The function of Lam16A in saccharification in cultured cells of U. esculenta and in cell wall depolymerization during smut gall formation in Z. latifolia infected with U. esculenta is also considered. MATERIALS AND METHODS Strains, culture conditions, and carbohydrates. U. esculenta (NBRC 9887) and A. oryzae (RIB-40) were obtained from the National Institute of Technology and Evaluation (NITE, Chiba, Japan). Z. latifolia infected with U. esculenta was gratefully provided by Y. Okubo. U. esculenta was grown in 1 liter of modified Czapek-Dox medium (3% sucrose, 24 mM NaNO3, 7.4 mM KH2PO4, 2.0 mM MgSO4, 6.7 mM KCl, 36 ␮M FeSO4 · 7H2O, and 10 ␮M thiamine [pH 6.5]) for 3 days at 25°C and at 130 rpm. Laminarin, carboxymethyl (CM)-cellulose, and hydroxyethyl (HE)cellulose were purchased from Sigma-Aldrich; CM-pachyman, CM-curdlan, lichenan, pachyman, barley 1,3-1,4-␤-glucan, tamarind xyloglucan, arabinogalactan, arabinan, and polygalacturonate were obtained from Megazyme (Wicklow, Ireland); and laminarioligosaccharides with a degree of polymerization of 2 to 7 were obtained from Seikagaku Biobusiness (Tokyo, Japan). Rice xylan was extracted from rice cell walls with 24% (wt/vol) KOH containing 0.05% (wt/vol) NaBH4 after treatment with 4% (wt/vol) KOH containing 0.05% NaBH4. Purification of laminarin-degrading enzymes. Culture filtrates of U. esculenta were collected through cheesecloth, equilibrated with 10 mM sodium phosphate buffer (pH 7.0), and loaded onto an anion-exchange column (MonoQ HR 5/5 [5 by 50 mm, 1 ml]; GE Healthcare, Buckinghamshire, United Kingdom). The unbound fraction eluted from the MonoQ column was equilibrated with 10 mM sodium phosphate buffer (pH 6.0) by ultrafiltration (Amicon Ultra-15; Millipore, MA) and loaded onto a weak-cation-exchange column (Toyopearl CM-650 [1 ml]; Tosoh, Tokyo, Japan) equilibrated with the same buffer. The unbound fraction eluted from the CM-650 column was equilibrated with 10 mM sodium acetate buffer (pH 4.0) by ultrafiltration and loaded onto a strong-cationexchange column (UNOsphere S [1 ml]; Bio-Rad, CA) equilibrated with the same buffer. After washing with the same buffer, bound proteins were eluted with a linear gradient of 0 to 0.05 M NaCl for 5 min and 0.05 to 0.25 M NaCl for 25 min in the same buffer at a flow rate of 1 ml/min. The eluate was collected in 1-ml portions. Assay for hydrolytic activity. The hydrolytic activity of the laminarindegrading enzyme was assayed by aniline blue staining during the purification procedure, as described previously (22). Reaction mixtures (25 ␮l) comprised of the enzyme fraction (5 ␮l), 0.1% (wt/vol) laminarin, and 100 mM sodium phosphate (pH 6.0) were incubated at 30°C for 1 h (weak-cation-exchange fractions) or 3.8 h (strong-cation-exchange fractions), after which 100 ␮l of aniline blue (0.033%, wt/vol) in 0.17 N HCl and 0.5 M glycine-NaOH (pH 9.5) were added. Residual laminarin was measured as the fluorescence (400-nm excitation and 480-nm emission) of the laminarin-aniline blue complex after a 30-min incubation at 50°C (SpectraMax 190 spectrophotometer; Molecular Devices, CA). The hydrolytic activity toward polysaccharides was measured by the increase in reducing ends using p-hydroxybenzoic acid hydrazide (PAHBAH) (24). A reaction mixture (40 ␮l) containing the enzyme preparation (0.063 ␮g), 0.02% (wt/vol) polysaccharide, 0.01% (wt/vol) bovine serum albumin (BSA), and 100 mM sodium acetate (pH 5.0) was incu-

August 2012 Volume 78 Number 16

bated at 30°C for 30 min. After centrifugation at 22,000 ⫻ g for 1 min, the supernatant (30 ␮l) was mixed with 90 ␮l of 1% (wt/vol) p-hydroxybenzoic acid hydrazide–HCl. The mixture was boiled for 5 min, and the absorbance was measured at 410 nm. The increase in reducing ends was calculated based on a glucose standard curve. Laminarioligosaccharide hydrolysis was assayed in the same way as for other polysaccharides except that the substrate concentration was 1 mM. The reaction was stopped by the incubation of the mixture at 80°C for 5 min. The reaction solution was diluted 10-fold with distilled water and analyzed by high-performance liquid chromatography (HPLC) with a Dionex ICS-3000 instrument (Dionex, CA) equipped with an anion-exchange column (CarboPac PA-1 [4 by 250 mm]; Dionex). Samples were eluted by using a gradient of 0 to 300 mM sodium acetate for 30 min in the presence of 100 mM NaOH at a flow rate of 0.5 ml/min. Activity was determined by the sum of released laminaribiose and laminaritriose. The quantification of peak areas corresponding to each sugar was based on standard calibration curves for laminarioligosaccharides (degree of polymerization of 2 to 7). Protein assay. Protein concentrations were determined by the bicinchoninic acid (BCA) method using BCA protein assay reagent (Thermo Fisher Scientific, MA). BSA was used as a standard protein. Total proteins and glycoproteins after SDS-PAGE were visualized by Sypro ruby and periodic acid-Schiff (PAS) staining, respectively, using a Pro-Q Emerald 300 glycoprotein gel stain kit (Invitrogen). Effect of temperature and pH on recombinant Lam16A-His7 activity. The effect of temperature on the hydrolytic activity of Lam16A-His7 was determined by performing the assay at 0°C to 60°C for 10 to 90 min with 100 mM sodium acetate buffer (pH 5.0) and laminarin as the substrate, followed by the PAHBAH assay. The effect of pH on activity was determined by incubation at 30°C for 30 min in the presence of sodium acetate (pH 3.5 to 5.0), morpholineethanesulfonic acid (MES)-NaOH (pH 5.0 to 6.0), or sodium phosphate (pH 6.0 to 7.0). The temperature and pH stability of Lam16A-His7 were determined as the activity after the preincubation of Lam16A-His7 (5.2 ␮g/ml) in 100 mM sodium acetate buffer (pH 5.0) containing 0.01% BSA at various temperatures for 1 h or after preincubation in sodium acetate (pH 3.5 to 5.0), MES-HCl (pH 5.0 to 6.0), sodium phosphate (pH 6.0 to 7.0), or Tris-HCl (pH 7.0 to 8.0) at 30°C for 1 h in the presence of 0.01% BSA. Kinetic analysis of Lam16A. Kinetic parameters of Lam16A (1.0 ␮g/ ml) on laminariheptaose (0.15 to 5.0 ␮M) were determined by regression analysis using KaleidaGraph, version 3.51, with the following equation, based on a Michaelis-Menten equation: v/[E0] ⫽ Km[S]/(Km ⫹ [S]), where v is the initial velocity of the production of laminaribiose and laminaritriose and [E0] is the enzyme concentration. DNA amplification and cloning. Genomic DNA was extracted from cultured U. esculenta cells by using a plant genome DNA extraction kit (G-Bioscience, MO). Total RNAs were prepared from U. esculenta and Z. latifolia by using an RNeasy plant mini kit (Qiagen, Hilden, Germany) and were treated with DNase I (Invitrogen, CA) for 15 min at 22°C. Firststrand cDNA was synthesized from total RNA with an oligo(dT)18 primer using SuperScriptIII reverse transcriptase (Invitrogen). PCRs were performed by using a reaction mixture containing PrimeStarGXL DNA polymerase (TaKaRa Bio, Shiga, Japan), PrimeStarGXL DNA polymerase buffer, 0.1 mM each deoxynucleoside triphosphate (dNTP), 0.3 ␮M primer pairs, and a DNA template. For the amplification of the 5= and 3= regions of lam16A, PCR was performed by using genomic DNA, primers 5=-GAGCTWSYTGSCTGCT GAKST-3= and 5=-GGCACCACSGGMAAGGGCGTCCGCGTKTGG-3= for the 5= region, and primers 5=-TGTACRGYKTGCAWYRTMC-3= and 5=-CCAMACGCGGACGCCCTTKCCSGTGGTGCC-3= for the 3= region (where W is A or T, S is C or G, Y is C or T, K is G or T, M is A or C, R is A or G, and M is A or C). Primers were designed based on the Sporisorium reilianum SRZ2 ␤-glucanase gene sequence. Primers 5=-GAAACACTTG ACGCATTCCGCCTCCTG-3= and 5=-GGTGGGGTTTGCATTGTCCA GAATCGC-3= were designed based on the 5= and 3= regions and were used

aem.asm.org 5683

Nakajima et al.

to amplify the complete open reading frame from U. esculenta cDNA pools. The amplified DNA was cloned into a pGEM-T Easy vector (Promega) and was used to transform Escherichia coli (DH5␣) cells by heat shock followed by selection on plates containing LB plus ampicillin (50 ␮g/ml). PCR products and plasmid constructs were sequenced by using a 3130 genetic analyzer (Applied Biosystems, CA). Sequence analysis. Conserved domains at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih .gov/) were searched. N-terminal signal sequences were predicted by the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and the NetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/NetOGlyc/) were used to predict glycosylation sites. GPI anchor signal sequences were predicted by the fungal big-II predictor (http://mendel.imp.univie.ac.at/gpi /fungi_server.html). SOSUI engine, version 1.10 (http://bp.nuap.nagoya -u.ac.jp/sosui/), was used for predictions of protein localization. Thirtysix amino acid sequences in GH16 were aligned by using ClustalW software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Overexpression of recombinant Lam16A. C-terminal His7-tagged lam16A (lam16A-His7) and N-terminal FLAG-tagged lam16A (lam16A-FLAG) fusions were generated by PCR using primers 5=-GT GGTGATGGCTAGGAGCCAGCGAAGCCATCACTGCAGCG-3= and 5=-TTAGTGATGGTGATGGTGGTGATGGCTAGGAGCCAGCG-3= or 5=-GGCCGCGCCCTCGCCGGCGACTATAAGGACGATGACGATAA GGCCAACTGGACACAGACCGCCGTC-3=, respectively, and were cloned into a pAmyB expression vector (47) linearized with NaeI by using an In-Fusion PCR cloning kit (Clontech, CA). A. oryzae was transformed with the plasmid as described previously (16, 47). Transformants were selected on Czapek-Dox plates supplemented with 0.1 mg/ml pyrithiamine and cultured in YPM medium containing 100 ␮g/ml ampicillin at 25°C for 3 days at 130 rpm, as described previously (41). Purification of recombinant Lam16A-His7. Culture filtrates obtained after 3 days of growth of the A. oryzae transformant expressing Lam16A-His7 were used to purify Lam16A-His7 by using polyhistidinebinding resin (Talon metal affinity resin; Clontech, CA) as described previously (42). Purified Lam16A-His7 was concentrated and equilibrated in 20 mM sodium phosphate buffer (pH 6.0) by ultrafiltration before use. Fractionation of recombinant Lam16A. A. oryzae transformants overexpressing Lam16A-FLAG or Lam16A⌬GPI-FLAG were cultured for 2 days in YPM medium at 25°C. The culture filtrate (40 ml) obtained following the filtration of cells through cheesecloth was concentrated to 500 ␮l by ultrafiltration and used as the extracellular fraction. The recombinant protein from the extracellular fraction was concentrated for immunoblot analysis as follows. Fractions (200 ␮l) were mixed with 50 ␮l of anti-FLAG M2 affinity gel (Sigma-Aldrich) equilibrated with 50 mM TrisHCl (pH 7.5) containing 150 mM NaCl on ice for 3 min. After three washes with 400 ␮l of the same buffer, bound proteins were eluted with 40 ␮l of a 2% (wt/vol) SDS solution. U. esculenta cells were homogenized with Lysing Matrix C (MP Biochemicals Tokyo, Japan) in 50 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl (buffer A), with vigorous shaking at 6 strokes s⫺1 in two 20-s pulses, and centrifuged at 7,400 ⫻ g for 5 min. The pellet, the cell wall fraction (700 mg of wet cells, equivalent to 70 ml of culture medium), was suspended in 3 ml of 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, and 2% (wt/vol) SDS and boiled for 10 min. The pellet was obtained by centrifugation at 5,000 ⫻ g. This procedure was repeated twice. The resulting pellet was washed five times with 100 mM sodium acetate (pH 5.5) containing 1 mM EDTA and treated with a mixture of Yatalase (5.4 mg, 30 U; TaKaRa Bio) and lysing enzyme (3 mg; Sigma-Aldrich) in 1.2 ml of 10 mM sodium phosphate (pH 6.0) containing 150 mM NaCl at 37°C for 2 h. The supernatant (100 ␮l) obtained after centrifugation for 5 min at 22,000 ⫻ g at 4°C was subjected to cold acetone precipitation. The precipitate obtained after centrifugation at 22,000 ⫻ g for 10 min was dissolved in 10 ␮l of SDS-PAGE sample buffer. The supernatant after the centrifugation of the homogenized cells described above

5684

aem.asm.org

was centrifuged at 22,000 ⫻ g for 15 min, and the pellet was collected as a membrane fraction. The membrane fraction was washed with buffer A three times, suspended in 300 ␮l of buffer A containing 1% (vol/vol) Triton X-100, and held for 1 h at 4°C. The supernatant obtained by centrifugation at 22,000 ⫻ g for 15 min was used as a membrane-solubilized fraction. The membranesolubilized fraction (100 ␮l) was subjected to cold-acetone precipitation. The pellet obtained by centrifugation at 22,000 ⫻ g for 5 min was air dried and dissolved in 20 ␮l of SDS-PAGE sample buffer before immunoblot analysis. Phosphatidylinositol-specific phospholipase C treatment of the membrane fraction. The membrane fraction was incubated in 100 ␮l of 50 mM Tris-HCl (pH 7.5) containing 5 mM EDTA and phosphatidylinositol-specific phospholipase C (PI-PLC) (0.4 U; Sigma-Aldrich) at 30°C for 1 h. Heat-inactivated PI-PLC (80°C for 5 min) was used as a control. Following the centrifugation of the reaction mixture at 22,000 ⫻ g for 10 min, the supernatant contained the PI-PLC-soluble fraction. The pellet was washed three times with 100 ␮l of buffer A and then incubated in 100 ␮l of buffer A containing 1% Triton X-100 on ice for 1 h. The supernatant obtained after the centrifugation of this solution at 22,000 ⫻ g for 10 min was precipitated with acetone, and the pellet was used as the PI-PLCinsoluble fraction for immunoblot analyses. Immunological analysis. Proteins were subjected to SDS-PAGE followed by blotting onto a membrane. The membrane was preincubated in PBST (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.1% [vol/vol] Tween 20) containing 1.5% (wt/vol) nonfat milk for 1 h at room temperature and then incubated for 1 h with a horseradish peroxidase-conjugated monoclonal antibody against the polyhistidine tag (Qiagen) or the FLAG tag (Sigma-Aldrich), diluted 1:10,000 in PBST. After the membrane was washed four times with PBST for 10 min, the antibody-antigen complex was detected by using an ECL advanced detection kit (GE Healthcare) and an LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Effect of Lam16A on glucose production from laminarin by UeBgl3A. Reaction mixtures (20 ␮l) containing Lam16A (0 to 2.0 ␮g), recombinant U. esculenta ␤-glucosidase (UeBgl3A) (0.02 ␮g) produced by A. oryzae (31), 0.1% (wt/vol) laminarin, and 100 mM sodium phosphate (pH 6.0) were incubated at 30°C for 30 min. The amount of glucose was determined by using a glucose oxidase assay kit (Megazyme) and was calculated based on a glucose calibration curve. RT-PCR of the lam16A gene. The extraction of total RNA from Z. latifolia galls at various stages (before hypertrophy and at hypertrophic stages of 1, 20, and 170 g of fresh weight) and first-strand cDNA synthesis were carried out as described above. Reverse transcription (RT)-PCR was performed by using primers 5=-ACTCGTCGCCATGGAACGATCTTTC GG-3= and 5=-GTTTGCGGTGCTACCACCAATAGTGTAG-3=. Actin DNA was amplified by PCR using primers 5=-GACGGACAGGTGATCA CCATTGGCAAC-3= and 5=-CTCCTGCTTCGAGATCCACATCTGCT G-3= to standardize reaction conditions. Nucleotide sequence accession number. The sequence of the gene encoding Lam16A has been deposited in the DNA Data Bank of Japan (DDBJ) under accession number AB691944.

RESULTS

Purification of laminarin-degrading enzyme. In a previous study, we reported that U. esculenta secreted enzymes responsible for laminarin degradation in culture medium containing glucose as the sole carbon source (32). In the present study, we investigated a protein from a U. esculenta culture filtrate with endotypic hydrolyzing activity on laminarin. When the culture filtrate was loaded onto an anion-exchange column, laminarin-degrading activity was detected in the unbound fraction. The application of the unbound fraction onto a Toyopearl CM-650 column, a weakcation-exchange column, also resulted in laminarin-degrading activity eluting in the unbound fraction (Fig. 1A). When the major

Applied and Environmental Microbiology

GPI-Anchored ␤-1,3-Glucanase from U. esculenta

FIG 1 Fractionation of enzymes with laminarin-hydrolyzing activity in the U. esculenta culture filtrate. Fractions obtained by weak-cation-exchange (A) and strong-cation-exchange (B) chromatography were subjected to SDS-PAGE followed by silver staining (top) and were then assayed for laminarin-degrading activity (bottom). M refers to protein size standards. Arrows indicate bands supplied for LC/MS/MS analysis (see the supplemental material).

enzymatically active fraction (fraction 2) (Fig. 1A) was loaded onto an UNOsphere S column, a strong-cation-exchange column, the major activity was detected in fractions 11 to 13, in which three major proteins of 60, 40, and 30 kDa were observed by silver staining (Fig. 1B). Identification of laminarin-degrading enzyme. The three major proteins were subjected to chymotrypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) (see the supplemental material). Three peptide sequences from the 60-kDa protein, two of which contained one mismatch each, were identical to those found in a putative GH16 ␤-1,3-glucanase from S. reilianum SRZ2 (Table 1). Two peptide sequences were obtained from the 40-kDa protein, one of which is annotated as a phosphatidylethanolamine-binding protein. The other showed no conserved domains based on a domain search at the NCBI website. One peptide sequence obtained from the 30kDa protein is not annotated as any conserved domain. Thus, it was concluded that the 60-kDa protein was responsible for laminarin degradation. Sequence analysis. The gene encoding the 60-kDa protein, the U. esculenta laminarin-degrading enzyme belonging to GH16 (Lam16A), was amplified by PCR from a U. esculenta cDNA pool using degenerate primers based on the S. reilianum SRZ2 GH16 ␤-glucanase gene. The cloned DNA consisted of 1,196 bp, including a predicted open reading frame of 1,173 bp. The translated amino acid sequence indicates that Lam16A contains the conserved GH16 catalytic domain, a secretion signal peptide consisting of 25 amino acids at the N terminus, and a GPI anchor signal peptide consisting of 29 amino acids at the C terminus. The N-terminal isoleucine in the amino acid sequence NRAGGGIIAMERSF from S. reilianum SRZ2 is replaced by leucine in Lam16A. This mismatch occurs because isoleucine and leucine have the same molecular weights. A phylogenetic tree of GH16 proteins shows that Lam16A belongs to the Basidiomycetes group, with a clear separation from Crh family proteins and characterized ␤-1,3-glucanases, largely from eukaryotes (see Fig. S1 in the supplemental material). Among Lam16A homologs, the GPI anchor signal pep-

August 2012 Volume 78 Number 16

tide is found only in proteins from the Ustilaginomycotina subphylum. Enzymatic properties of recombinant Lam16A-His7. In order to generate recombinant Lam16A, 5 C-terminal amino acid residues from Lam16A were replaced by heptahistidine residues (His7), because native Lam16A fused with C-terminal His7 could not be achieved (data not shown). Recombinant Lam16A-His7 overexpressed in A. oryzae was purified by using polyhistidine affinity chromatography before enzymatic properties were determined. Purified Lam16A-His7 exhibited a single broad band at around 110 kDa by Sypro ruby staining (see Fig. S2A in the supplemental material). Immunoblot analysis using an antibody against polyhistidine showed a single band with a molecular mass identical to that observed by Sypro ruby staining (see Fig. S2B in the supplemental material). The enzyme was detected clearly by glycoprotein analysis (see Fig. S2C in the supplemental material), indicating that recombinant Lam16A-His7 was highly glycosylated, because Lam16A-His7 has a calculated molecular mass of 39 kDa, with 7 potential N-glycosylation sites and 9 potential O-glycosylation sites. The maximum hydrolytic activity on laminarin was observed at pH 5.0 and at 40°C (Fig. 2A and B). The enzyme retained over 80% residual activity after incubation at 10°C to 40°C or pH 3.5 to 7.0 for 1 h (Fig. 2C and D). Substrate specificity. To investigate substrate specificity, the hydrolytic activity toward polysaccharides was determined; among the polysaccharides tested, Lam16A-His7 exhibited activity only on ␤-1,3-glucan (Table 2), indicating that the enzyme is highly specific for ␤-1,3-glucan hydrolysis. The activity toward laminarioligosaccharides showed that Lam16A-His7 preferentially hydrolyzed laminarioligosaccharides with a degree of polymerization of ⬎4 (Table 2). Lam16A exhibited the highest activity toward laminariheptaose among the substrates tested, showing a Km of 0.65 ⫾ 0.21 mM and a kcat of 21 ⫾ 2.2 s⫺1. The end products generated from the hydrolysis of laminarioligosaccharides were laminaribiose and laminaritriose, and transglycosylation activity toward laminarioligosaccharides was not observed (data not shown). Effect of the GPI anchor signal peptide on localization of Lam16A-FLAG. The Lam16A localization in an A. oryzae transformant overexpressing Lam16A-FLAG or Lam16A⌬GPI-FLAG was determined immunologically in order to investigate the role of the putative GPI anchor signal peptide. Lam16A-FLAG was found in the membrane and extracellular fractions but not in the cell wall fraction, whereas Lam16A⌬GPI-FLAG was detected only in the extracellular fraction (Fig. 3A). Similarly, higher hydrolytic activity toward laminarin was detected in the membrane fraction

TABLE 1 Peptide sequences obtained by LC/MS/MS analysis

Peptide sequencea

Species

GTTGKGVRVW

S. reilianum SRZ2 S. reilianum SRZ2 S. reilianum SRZ2

NRAGGGIIAMERSF NQSGCNAQYPACSY a

DDBJ accession no. CBQ72681.1 CBQ72681.1 CBQ72681.1

Annotation GH16 ␤-glucanase GH16 ␤-glucanase GH16 ␤-glucanase

Underlining indicates a mismatched amino acid.

aem.asm.org 5685

Nakajima et al.

FIG 2 Effect of temperature and pH on hydrolytic activity of recombinant Lam16A-His7. The hydrolytic activity of Lam16A toward laminarin was assayed under various conditions. (A) The optimal temperature was determined after reaction mixtures containing laminarin, sodium acetate buffer (100 mM; pH 5.0), and Lam16A (0.02 ␮g) were incubated for 10 to 90 min at 10°C to 60°C. (B) The optimal pH was determined by incubation with sodium acetate (pH 4.0 to 5.0), MES-NaOH (pH 5.0 to 6.0), or sodium phosphate (pH 6.0 to 7.0) at 30°C for 30 min. (C and D) Temperature (C) and pH (B) stability for Lam16A was determined by assaying the hydrolytic activity after incubation at the indicated temperatures or with buffer (sodium acetate [pH 3.5 to 5.0] MES-NaOH [pH 5.0 to 6.0], sodium phosphate [pH 6.0 to 7.0], and Tris-HCl [pH 7.0 to 8.0]) for 1 h. Data are means of data from 3 individual determinations ⫾ standard errors.

from the transformant overexpressing Lam16A-FLAG and in the extracellular fraction from the transformant overexpressing Lam16A⌬GPI-FLAG (Fig. 3B and C). The results from the enzyme assay are consistent with those from the immunoblot analysis. These results indicate that the GPI anchor signal peptide plays a significant role in the localization of Lam16A in the membrane. Effect of PI-PLC treatment on localization of Lam16AFLAG. The effect of PI-PLC treatment on the localization of Lam16A-FLAG was examined. PI-PLC treatment resulted in

Lam16A-FLAG localizing in the PI-PLC-soluble fraction, whereas the enzyme remained in the membrane fraction after treatment with heat-inactivated PI-PLC (Fig. 4). Immunoblot analysis revealed two and three bands representing Lam16A-FLAG, likely due to a variable degree of glycosylation. These results imply that the digestion of the GPI anchor with PI-PLC released Lam16A from the membrane. Enhanced glucose production by the action of Lam16A. Glucose production from laminarin using purified Lam16A and

TABLE 2 Substrate specificity of Lam16Aa Substrate

Mean sp act (U/mg) ⫾ SE

Relative activity (%)

Laminarin, 0.02% Laminaribiose Laminaritriose Laminaritetraose Laminaripentaose Laminarihexaose Laminariheptaose CM-curdlan, 0.02% CM-pachyman, 0.02%

6.2 ⫾ 0.28 ND ND 0.95 ⫾ 0.11 5.7 ⫾ 0.56 12 ⫾ 0.27 16 ⫾ 2.4 10 ⫾ 0.083 4.1 ⫾ 0.17

100

15 92 190 260 165 66

a ND indicates that the activity was less than 0.1 U/mg of specific activity. The specific activity of Lam16A toward lichenan, barley ␤-glucan, CM-cellulose, HE-cellulose, tamarind xyloglucan, arabinan, arabinogalactan, polygalacturonate, xylan, or pachyman as the substrate was less than 0.1 U/mg.

5686

aem.asm.org

FIG 3 Effect of the GPI anchor signal peptide on localization of Lam16A. (A) Immunoblot analysis of Lam16A-FLAG (GPI) and Lam16A⌬GPI-FLAG (⌬GPI). E, M, and C represent extracellular, membrane, and cell wall fractions, respectively. Each fraction is equivalent to 6 ml of culture. (B and C) The hydrolytic activity of the membrane (B) and extracellular (C) fractions was determined by using laminarin as a substrate.

Applied and Environmental Microbiology

GPI-Anchored ␤-1,3-Glucanase from U. esculenta

FIG 4 Effect of PI-PLC treatment on membrane-bound Lam16A-FLAG. ⫹ and ⫺ represent active and inactivated PI-PLC, respectively. The top and bottom columns represent PI-PLC-soluble and insoluble fractions, respectively.

UeBgl3A was assayed (Fig. 5). UeBgl3A produced 1.78 ␮g glucose from laminarin in this experiment, whereas Lam16A (2.0 ␮g) released only 0.64 ␮g glucose. The levels of glucose production were increased as the amount of added Lam16A increased. Maximal glucose production (6.94 ␮g) was attained in the presence of 1.0 ␮g Lam16A, about 4-fold higher than that produced by UeBgl3A only. This result suggests that the production of laminarioligosaccharides from laminarin by the action of Lam16A enhanced glucose production by UeBgl3A. The expression levels of lam16A and UeBgl3A were examined by PCR to investigate their involvement during Z. latifolia gall formation. High levels of lam16A transcripts were detected at the initial stage of gall formation (Fig. 6), and UeBgl3A was expressed constitutively. This result may suggest that Lam16A is involved in cell wall loosening, which leads to massive cell expansion, and in metabolizing ␤-1,3-glucan with the cooperative action of UeBgl3A. DISCUSSION

U. esculenta cells grown in liquid medium secrete an enzyme involved in laminarin degradation. Lam16A was able to degrade ␤-1,3-glucan specifically and preferred substrates with a degree of polymerization of ⬎4. UeBgl3A, a ␤-glucosidase from U. esculenta, produces glucose from a variety of ␤-glucosides. Laminarioligosaccharides with degrees of polymerization of 2 to 4 have been reported to be substrates that are easily hydrolyzed by this enzyme (32). The cooperative activity of Lam16A and UeBgl3A is suggested to be involved in the digestion of ␤-1,3-glucan to glucose efficiently during the growth of U. esculenta in culture medium and in Z. latifolia (Fig. 5 and 6).

FIG 5 Effect of Lam16A on glucose production by UeBgl3A. The amount of glucose released from laminarin by UeBgl3A (0.02 ␮g) was determined in the presence of Lam16A (0 to 2.0 ␮g).

August 2012 Volume 78 Number 16

FIG 6 Gene expression analysis of lam16A during Z. latifolia gall formation. (A) Z. latifolia galls at various hypertrophic stages were used for RT-PCR. Lane 1, stage before hypertrophy; lanes 2, 3, and 4, 1 g, 20 g, and 170 g gall fresh weight of hypertrophic stage, respectively. Bars represent 5 cm. (B) Transcript levels of lam16A and UeBgl3A were analyzed by RT-PCR. Amplified DNA fragments were stained with ethidium bromide.

Lam16A was partially purified from the culture filtrate, indicating that the enzyme is secreted extracellularly. However, amino acid sequence analysis of Lam16A revealed a C-terminal GPI anchor signal peptide that indicates a plasma membrane location. Recent proteomic analyses have shown that many proteins possessing GPI anchor signal peptides from Aspergillus nidulans and S. cerevisiae are covalently linked to the cell wall (6, 12, 14, 15, 17, 48), suggesting a possible cell wall location for Lam16A. While computational predictions would be helpful, to our knowledge, systematic sequence-based predictions of cell wall localization have not yet been performed for proteins in any organism. To investigate the function of a possible GPI anchor signal peptide in Lam16A, the localization of recombinant Lam16A-FLAG and Lam16A⌬GPI-FLAG in A. oryzae was detected immunologically. Most of the Lam16A-FLAG protein was found in the membrane and extracellular fractions (Fig. 3A). Conversely, Lam16A⌬GPIFLAG, lacking the GPI anchor signal peptide, was found only in the extracellular fraction. Furthermore, the treatment of the membrane fraction from A. oryzae with PI-PLC released Lam16AFLAG into the PI-PLC-soluble fraction (Fig. 4). These results led to the conclusion that the GPI anchor signal peptide in Lam16A plays a significant role in its membrane localization with the GPI anchor. GPI-anchored proteins in A. fumigatus have been reported to be released from membrane preparations by endogenous PI-PLC (4). Similarly, Lam16A found in extracellular fractions from U. esculenta and A. oryzae might be released by the action of endogenous PI-PLC. The proteolytic release of Lam16A might also occur by analogy to yeast Crh2p, which is released from the membrane depending on the activity of the transmembrane protease Yps1p (30). The hydrolytic activity of the membrane fraction from A. oryzae overexpressing Lam16A-FLAG was much higher than that of the membrane fraction from the Lam16A⌬GPI-FLAG-overexpressing strain (Fig. 3B), indicating that Lam16A in the membrane is enzymatically active. Based on these results, we propose that Lam16A is transferred to the plasma membrane, where the enzyme hydrolyzes ␤-1,3-glucan, and that the enzyme is used even after it is released extracellularly.

aem.asm.org 5687

Nakajima et al.

Lam16A-His7 was found to hydrolyze ␤-1,3-glucan specifically and to act most efficiently on substrates with a degree of polymerization of ⬎4 (Table 2). This suggests cooperative activity with UeBgl3A, which acts preferentially on laminarioligosaccharides with a degree of polymerization of 2 to 4 to produce glucose. Lam16A did not exhibit transglycosylation activity toward laminarioligosaccharides, unlike Eng2, possessing a GPI anchor signal peptide, a GH16 endo-␤-1,3(4)-glucanase with transglycosylation activity toward laminaritetraose (18), or Crh family proteins (7). Such a range of activity may have evolved by a process of accumulated DNA substitutions, as seen in the diversity of amino acid sequences in the GH16 phylogenetic tree (see Fig. S1 in the supplemental material), in which the GPI anchor signal is conserved only in the Ustilaginomycotina subphylum among the Basidiomycetes cluster. Together, these results suggest that GPI-anchored Lam16A modifies the inner surface of the endogenous cell wall and afterwards is secreted to cleave ␤-1,3-glucan randomly to loosen the cell wall of the pathogen and/or the host, allowing for the catabolic use of ␤-1,3-glucan through the cooperative action of ␤-glucosidases. This possibility is supported by previously reported observations that Utr2, Crh11, and Crh12, all GPI-anchored cell wall proteins, play significant roles in cell wall formation (6, 34), and pea cellulase localizes at the inner surface of cell wall in auxintreated pea epicotyl (2). Furthermore, as ␤-1,3-glucan and its sulfated derivative form become elicitor molecules that induce defense responses in various plants, their catabolism would play an important additional role in depressing the activation of host defense mechanisms (1, 20, 21, 28). During gall formation, Lam16A could function to modify the U. esculenta cell wall and to degrade plant ␤-1,3-glucan (callose) in the host cell wall. The synthesis and degradation of callose are involved in plant growth and development, plasmodesma regulation, and the stress response (11), even though callose is only a minor cell wall component. The action of Lam16A may also cause morphological changes, especially during gall formation in Z. latifolia, when lam16A is highly expressed. We anticipate that the findings reported here on the localization and hydrolytic activity of a novel GPI-anchored ␤-1,3-glucanase will lead to a greater understanding of the significance of cell wall modifications by hydrolytic enzymes during hyphal extension and fungal growth and their effects on host plant morphogenesis.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22. 23. 24. 25.

ACKNOWLEDGMENTS We thank R. Oba, M. Kikuchi, and M. Iwai for technical assistance in preparing plasmid DNA and transforming A. oryzae. This work was supported in part by grant no. 23780111 from the Japanese Society for the Promotion of Science.

26.

REFERENCES

28.

1. Aziz A, et al. 2003. Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol. Plant Microbe Interact. 16:1118 –1128. 2. Bal AK, Verma DPS, Byrne H, Maclachlan G. 1976. Subcellular localization of cellulases in auxin-treated pea. J. Cell Biol. 69:97–105. 3. Bhatnagar RS, Gordon JI. 1997. Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol. 7:14 –20. 4. Bruneau JM, et al. 2001. Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis 22:2812–2823. 5. Bulow R, Nonnengasser C, Overath P. 1989. Release of the variant

5688

aem.asm.org

27.

29.

30.

31.

surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 32:85–92. Cabib E, Blanco N, Grau C, Rodriguez-Pena JM, Arroyo J. 2007. Crh1p and Crh2p are required for the cross-linking of chitin to ␤(1-6)glucan in the Saccharomyces cerevisiae cell wall. Mol. Microbiol. 63:921–935. Cabib E, et al. 2008. Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro. J. Biol. Chem. 283:29859 –29872. Carpita NC, Gibeaut DM. 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3:1–30. Chan TS, Thrower LB. 1980. The host-parasite relationship between Zizania caduciflora Turcz. and Ustilago esculenta P. Henn., II. Ustilago esculenta in culture. New Phytol. 85:209 –216. Chatterjee S, Mayor S. 2001. The GPI-anchor and protein sorting. Cell. Mol. Life Sci. 58:1969 –1987. Chen XY, Kim JY. 2009. Callose synthesis in higher plants. Plant Signal. Behav. 4:489 – 492. de Groot PW, et al. 2009. Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans. Fungal Genet. Biol. 46:S72–S81. doi: 10.1016/j.fgb.2008.07.022. de Groot PW, Hellingwerf KJ, Klis FM. 2003. Genome-wide identification of fungal GPI proteins. Yeast 20:781–796. Fujii T, Shimoi H, Iimura Y. 1999. Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1427:133–144. Fujita M, Jigami Y. 2008. Lipid remodeling of GPI-anchored proteins and its function. Biochim. Biophys. Acta 1780:410 – 420. Gomi K, Imura Y, Hara S. 1987. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51:2549 –2555. Hamada K, Fukuchi S, Arisawa M, Baba M, Kitada K. 1998. Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae. Mol. Gen. Genet. 258:53–59. Hartl L, Gastebois A, Aimanianda V, Latge JP. 2011. Characterization of the GPI-anchored endo ␤-1,3-glucanase Eng2 of Aspergillus fumigatus. Fungal Genet. Biol. 48:185–191. Hartland RP, et al. 1996. A novel ␤-(1-3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J. Biol. Chem. 271:26843–26849. Inui H, Yamaguchi Y, Hirano S. 1997. Elicitor actions of Nacetylchitooligosaccharides and laminarioligosaccharides for chitinase and L-phenylalanine ammonia-lyase induction in rice suspension culture. Biosci. Biotechnol. Biochem. 61:975–978. Klarzynski O, et al. 2000. Linear ␤-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiol. 124:1027–1038. Ko YT, Cheng WY. 2005. Fluorescence versus radioactivity for assaying antifungal compound inhibited yeast 1,3-␤-glucan synthase activity. J. Food Drug Anal. 13:184 –191. Lemansky P, et al. 1990. Dynamics and longevity of the glycolipidanchored membrane protein, Thy-1. J. Cell Biol. 110:1525–1531. Lever M. 1972. A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47:273–279. McConville MJ, Ferguson MA. 1993. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem. J. 294:305–324. McLaughlin S, Aderem A. 1995. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20:272–276. McQueen-Mason S, Durachko DM, Cosgrove DJ. 1992. Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4:1425– 1433. Menard R, et al. 2004. ␤-1,3-Glucan sulfate, but not ␤-1,3-glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis. Plant Cell 16:3020 –3032. Michel G, Tonon T, Scornet D, Cock JM, Kloareg B. 2010. Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in eukaryotes. New Phytol. 188:67– 81. Miller KA, DiDone L, Krysan DJ. 2010. Extracellular secretion of overexpressed glycosylphosphatidylinositol-linked cell wall protein Utr2/ Crh2p as a novel protein quality control mechanism in Saccharomyces cerevisiae. Eukaryot. Cell 9:1669 –1679. Mills K, et al. 2001. Identification of ␣1-antitrypsin variants in plasma with the use of proteomic technology. Clin. Chem. 47:2012–2022.

Applied and Environmental Microbiology

GPI-Anchored ␤-1,3-Glucanase from U. esculenta

32. Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T. 2012. Identification, cloning, and characterization of ␤-glucosidase from Ustilago esculenta. Appl. Microbiol. Biotechnol. 93:1989 –1998. 33. Orlean P, Menon AK. 2007. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids. J. Lipid Res. 48:993–1011. 34. Pardini G, et al. 2006. The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans. J. Biol. Chem. 281:40399 – 40411. 35. Pitson SM, Seviour RJ, McDougall BM. 1993. Noncellulolytic fungal ␤-glucanases: their physiology and regulation. Enzyme Microb. Technol. 15:178 –192. 36. Ragni E, Fontaine T, Gissi C, Latge JP, Popolo L. 2007. The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis. Yeast 24:297–308. 37. Ruel K, Joseleau JP. 1991. Involvement of an extracellular glucan sheath during degradation of populus wood by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 57:374 –384. 38. Seyfang A, Mecke D, Duszenko M. 1990. Degradation, recycling, and shedding of Trypanosoma brucei variant surface glycoprotein. J. Protozool. 37:546 –552. 39. Shukla SD, Coleman R, Finean JB, Michell RH. 1980. Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C. Biochem. J. 187:277–280. 40. Simons K, Ikonen E. 1997. Functional rafts in cell membranes. Nature 387:569 –572.

August 2012 Volume 78 Number 16

41. Takahashi M, et al. 2010. Characterization of a cellobiohydrolase (MoCel6A) produced by Magnaporthe oryzae. Appl. Environ. Microbiol. 76:6583– 6590. 42. Takeda T, et al. 2010. Characterization of endo-1,3-1,4-␤-glucanases in GH family 12 from Magnaporthe oryzae. Appl. Microbiol. Biotechnol. 88:1113–1123. 43. Takeda T, et al. 2002. Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments. Proc. Natl. Acad. Sci. U. S. A. 99:9055–9060. 44. Vasur J, et al. 2009. X-ray crystal structures of Phanerochaete chrysosporium laminarinase 16A in complex with products from lichenin and laminarin hydrolysis. FEBS J. 276:3858 –3869. 45. Yamazaki H, Tanaka A, Kaneko J, Ohta A, Horiuchi H. 2008. Aspergillus nidulans ChiA is a glycosylphosphatidylinositol (GPI)-anchored chitinase specifically localized at polarized growth sites. Fungal Genet. Biol. 45:963– 972. 46. Yang HC, Leu LS. 1978. Formation and histopathology of galls induced by Ustilago esculenta in Zizania latifolia. Phytopathology 68: 1572–1576. 47. Yano A, Kikuchi S, Nakagawa Y, Sakamoto Y, Sato T. 2009. Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae. Microbiol. Res. 164:642– 649. 48. Yin QY, et al. 2005. Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages. J. Biol. Chem. 280:20894 –20901.

aem.asm.org 5689