A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolasefrom 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 esculentaculture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzy-matically 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 �-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianumSRZ2 �-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal gly-cosylphosphatidylinositol (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 theextracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide wassecreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released com-pletely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in theplasma membrane in order to modify �-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catab-olism of �-1,3-glucan after its release in the extracellular medium.

�-1,3-Glucan occurs widely as a major component of the cellwalls 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-glu-can, called callose, in response to biotic or abiotic stress (11), whilesome algae accumulate �-1,3-glucan as a storage polysaccharide(29).

Several glycoside hydrolases are involved in �-1,3-glucan bio-synthesis and degradation. The hydrolysis of �-1,3-glucan can beperformed by the combined action of endo-�-1,3-glucanases aswell as �-glucosidases or exo-�-1,3-glucanases, which are foundin 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-Gluca-nosyltransferase from Aspergillus fumigatus, a member of GH72,catalyzes the hydrolysis of �-1,3-glucan and simultaneously pro-duces an insoluble �-1,3-glucan from laminarioligosaccharides bya transglycosylation reaction (19). The modification of �-1,3-glu-can by these enzymes is thought to play a significant role in cellwall morphogenesis and in nutrient (carbon) acquisition.

Glycosylphosphatidylinositol (GPI) anchoring is a posttrans-lational lipid modification that anchors proteins to the plasmamembrane. A number of examples are known, including a varietyof glycoside hydrolases, such as endo-�-1,3-glucanase and chiti-nase in GH16, GH17, GH72, and GH81 (13, 36, 45), and proteinsinvolved in transmembrane signaling, e.g., receptors and adhe-sion molecules (3, 26, 33). The core structure of the GPI anchormoiety is highly conserved from yeast to mammals (39). GPI an-choring to proteins occurs in the endoplasmic reticulum. GPI-anchored proteins then transit the secretory pathway to reach thecell surface (33). The GPI anchor moiety is further modified dur-ing transport. The remodeling process is essential for the properassociation of GPI-anchored proteins with lipid microdomains(lipid rafts), areas rich in sphingolipids and sterols formed by

localized phase separation in the plasma membrane (10, 40). GPI-anchored proteins bound to the membrane have a longer turnovertime than membrane proteins with a proteinaceous transmem-brane anchor (5, 23, 38). Side chains in the GPI anchor moietyallow for a high packing density of the tethered proteins (25).

Proteomic analyses have revealed that many proteins that pos-sess a GPI anchor signal peptide are covalently linked to the cellwall (6, 12, 14, 15, 25, 48). Crh1p and Crh2p, which are putativeGPI-anchored enzymes belonging to GH16, are covalently linkedto the cell wall of Saccharomyces cerevisiae and catalyze the trans-glycosylation of chitin to �-1,3-glucose branches of the �-1,6-glucan backbone (7). The deletion of genes encoding Crh familyproteins caused a remarkable reduction in the amount of �-1,6-glucan in the cell wall of Candida albicans (34). These observationssuggest the involvement of Crh1p and Crh2p in cell wall organi-zation. Thus, GPI-anchored glucoside hydrolases are found to lo-calize to the plasma membrane or cell wall through a GPI anchormoiety. However, the significance of the hydrolytic activity ofGPI-anchored glycoside hydrolases remains unknown. In thisstudy, we identify and characterize for the first time, to our knowl-edge, a GPI-anchored �-1,3-glucanase (Lam16A) from Ustilagoesculenta that is localized to the membrane via a GPI anchor butthat is also secreted extracellularly.

The basidiomycetous fungus Ustilago esculenta is infectious to

Received 16 February 2012 Accepted 25 May 2012

Published ahead of print 8 June 2012

Address correspondence to Takumi Takeda, ttakeda@ibrc.or.jp.

* 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

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Zizania latifolia (aquatic grass) and causes smut gall in the flow-ering 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. esculentahave been proposed to be involved in the depolymerization of cellwall polysaccharides, which induces the elongation of hyphae andresults in host cell wall degradation and loosening during plantcell enlargement (8, 27, 43). The action of endo-�-1,3-glucanasecould contribute to the cell wall modification of the fungus and ofthe plant host as well as to saccharification in concert with �-glu-cosidases (32). The function of Lam16A in saccharification in cul-tured cells of U. esculenta and in cell wall depolymerization duringsmut gall formation in Z. latifolia infected with U. esculenta is alsoconsidered.

MATERIALS AND METHODSStrains, culture conditions, and carbohydrates. U. esculenta (NBRC9887) and A. oryzae (RIB-40) were obtained from the National Institute ofTechnology and Evaluation (NITE, Chiba, Japan). Z. latifolia infectedwith U. esculenta was gratefully provided by Y. Okubo. U. esculenta wasgrown in 1 liter of modified Czapek-Dox medium (3% sucrose, 24 mMNaNO3, 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-curd-lan, lichenan, pachyman, barley 1,3-1,4-�-glucan, tamarind xyloglucan,arabinogalactan, arabinan, and polygalacturonate were obtained fromMegazyme (Wicklow, Ireland); and laminarioligosaccharides with a de-gree of polymerization of 2 to 7 were obtained from Seikagaku Biobusi-ness (Tokyo, Japan). Rice xylan was extracted from rice cell walls with24% (wt/vol) KOH containing 0.05% (wt/vol) NaBH4 after treatmentwith 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 mMsodium phosphate buffer (pH 7.0), and loaded onto an anion-exchangecolumn (MonoQ HR 5/5 [5 by 50 mm, 1 ml]; GE Healthcare, Bucking-hamshire, United Kingdom). The unbound fraction eluted from theMonoQ column was equilibrated with 10 mM sodium phosphate buffer(pH 6.0) by ultrafiltration (Amicon Ultra-15; Millipore, MA) and loadedonto a weak-cation-exchange column (Toyopearl CM-650 [1 ml]; Tosoh,Tokyo, Japan) equilibrated with the same buffer. The unbound fractioneluted from the CM-650 column was equilibrated with 10 mM sodiumacetate buffer (pH 4.0) by ultrafiltration and loaded onto a strong-cation-exchange column (UNOsphere S [1 ml]; Bio-Rad, CA) equilibrated withthe same buffer. After washing with the same buffer, bound proteins wereeluted with a linear gradient of 0 to 0.05 M NaCl for 5 min and 0.05 to 0.25M NaCl for 25 min in the same buffer at a flow rate of 1 ml/min. The eluatewas collected in 1-ml portions.

Assay for hydrolytic activity. The hydrolytic activity of the laminarin-degrading enzyme was assayed by aniline blue staining during the purifi-cation procedure, as described previously (22). Reaction mixtures (25 �l)comprised of the enzyme fraction (5 �l), 0.1% (wt/vol) laminarin, and100 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 frac-tions), after which 100 �l of aniline blue (0.033%, wt/vol) in 0.17 N HCland 0.5 M glycine-NaOH (pH 9.5) were added. Residual laminarin wasmeasured 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 bythe increase in reducing ends using p-hydroxybenzoic acid hydrazide(PAHBAH) (24). A reaction mixture (40 �l) containing the enzyme prep-aration (0.063 �g), 0.02% (wt/vol) polysaccharide, 0.01% (wt/vol) bovineserum albumin (BSA), and 100 mM sodium acetate (pH 5.0) was incu-

bated at 30°C for 30 min. After centrifugation at 22,000 � g for 1 min, thesupernatant (30 �l) was mixed with 90 �l of 1% (wt/vol) p-hydroxyben-zoic acid hydrazide–HCl. The mixture was boiled for 5 min, and the ab-sorbance was measured at 410 nm. The increase in reducing ends wascalculated based on a glucose standard curve.

Laminarioligosaccharide hydrolysis was assayed in the same way as forother polysaccharides except that the substrate concentration was 1 mM.The reaction was stopped by the incubation of the mixture at 80°C for 5min. The reaction solution was diluted 10-fold with distilled water andanalyzed by high-performance liquid chromatography (HPLC) with aDionex ICS-3000 instrument (Dionex, CA) equipped with an anion-ex-change column (CarboPac PA-1 [4 by 250 mm]; Dionex). Samples wereeluted by using a gradient of 0 to 300 mM sodium acetate for 30 min in thepresence of 100 mM NaOH at a flow rate of 0.5 ml/min. Activity wasdetermined by the sum of released laminaribiose and laminaritriose. Thequantification of peak areas corresponding to each sugar was based onstandard calibration curves for laminarioligosaccharides (degree of po-lymerization of 2 to 7).

Protein assay. Protein concentrations were determined by the bicin-choninic acid (BCA) method using BCA protein assay reagent (ThermoFisher Scientific, MA). BSA was used as a standard protein. Total proteinsand glycoproteins after SDS-PAGE were visualized by Sypro ruby andperiodic acid-Schiff (PAS) staining, respectively, using a Pro-Q Emerald300 glycoprotein gel stain kit (Invitrogen).

Effect of temperature and pH on recombinant Lam16A-His7 activ-ity. 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 minwith 100 mM sodium acetate buffer (pH 5.0) and laminarin as the sub-strate, followed by the PAHBAH assay. The effect of pH on activity wasdetermined by incubation at 30°C for 30 min in the presence of sodiumacetate (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 temperatureand pH stability of Lam16A-His7 were determined as the activity after thepreincubation of Lam16A-His7 (5.2 �g/ml) in 100 mM sodium acetatebuffer (pH 5.0) containing 0.01% BSA at various temperatures for 1 h orafter preincubation in sodium acetate (pH 3.5 to 5.0), MES-HCl (pH 5.0to 6.0), sodium phosphate (pH 6.0 to 7.0), or Tris-HCl (pH 7.0 to 8.0) at30°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 regressionanalysis 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 lami-naritriose and [E0] is the enzyme concentration.

DNA amplification and cloning. Genomic DNA was extracted fromcultured 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. First-strand cDNA was synthesized from total RNA with an oligo(dT)18 primerusing SuperScriptIII reverse transcriptase (Invitrogen). PCRs were per-formed by using a reaction mixture containing PrimeStarGXL DNA poly-merase (TaKaRa Bio, Shiga, Japan), PrimeStarGXL DNA polymerase buf-fer, 0.1 mM each deoxynucleoside triphosphate (dNTP), 0.3 �M primerpairs, and a DNA template.

For the amplification of the 5= and 3= regions of lam16A, PCR wasperformed by using genomic DNA, primers 5=-GAGCTWSYTGSCTGCTGAKST-3= and 5=-GGCACCACSGGMAAGGGCGTCCGCGTKTGG-3=for the 5= region, and primers 5=-TGTACRGYKTGCAWYRTMC-3= and5=-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 isA or G, and M is A or C). Primers were designed based on the Sporisoriumreilianum SRZ2 �-glucanase gene sequence. Primers 5=-GAAACACTTGACGCATTCCGCCTCCTG-3= and 5=-GGTGGGGTTTGCATTGTCCAGAATCGC-3= were designed based on the 5= and 3= regions and were used

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to amplify the complete open reading frame from U. esculenta cDNApools. The amplified DNA was cloned into a pGEM-T Easy vector (Pro-mega) and was used to transform Escherichia coli (DH5�) cells by heatshock followed by selection on plates containing LB plus ampicillin (50�g/ml). PCR products and plasmid constructs were sequenced by using a3130 genetic analyzer (Applied Biosystems, CA).

Sequence analysis. Conserved domains at the National Center forBiotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) were searched. N-terminal signal sequences were predicted by theSignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The Net-NGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and theNetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/NetOGlyc/) wereused to predict glycosylation sites. GPI anchor signal sequences were pre-dicted 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. Thirty-six amino acid sequences in GH16 were aligned by using ClustalWsoftware (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Overexpression of recombinant Lam16A. C-terminal His7-taggedlam16A (lam16A-His7) and N-terminal FLAG-tagged lam16A(lam16A-FLAG) fusions were generated by PCR using primers 5=-GTGGTGATGGCTAGGAGCCAGCGAAGCCATCACTGCAGCG-3= and5=-TTAGTGATGGTGATGGTGGTGATGGCTAGGAGCCAGCG-3= or5=-GGCCGCGCCCTCGCCGGCGACTATAAGGACGATGACGATAAGGCCAACTGGACACAGACCGCCGTC-3=, respectively, and werecloned into a pAmyB expression vector (47) linearized with NaeI by usingan In-Fusion PCR cloning kit (Clontech, CA). A. oryzae was transformedwith the plasmid as described previously (16, 47). Transformants wereselected on Czapek-Dox plates supplemented with 0.1 mg/ml pyrithia-mine and cultured in YPM medium containing 100 �g/ml ampicillin at25°C for 3 days at 130 rpm, as described previously (41).

Purification of recombinant Lam16A-His7. Culture filtrates ob-tained after 3 days of growth of the A. oryzae transformant expressingLam16A-His7 were used to purify Lam16A-His7 by using polyhistidine-binding resin (Talon metal affinity resin; Clontech, CA) as described pre-viously (42). Purified Lam16A-His7 was concentrated and equilibrated in20 mM sodium phosphate buffer (pH 6.0) by ultrafiltration before use.

Fractionation of recombinant Lam16A. A. oryzae transformantsoverexpressing Lam16A-FLAG or Lam16A�GPI-FLAG were cultured for2 days in YPM medium at 25°C. The culture filtrate (40 ml) obtainedfollowing the filtration of cells through cheesecloth was concentrated to500 �l by ultrafiltration and used as the extracellular fraction. The recom-binant protein from the extracellular fraction was concentrated for im-munoblot analysis as follows. Fractions (200 �l) were mixed with 50 �l ofanti-FLAG M2 affinity gel (Sigma-Aldrich) equilibrated with 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl on ice for 3 min. After threewashes 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 Bio-chemicals 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, thecell wall fraction (700 mg of wet cells, equivalent to 70 ml of culturemedium), was suspended in 3 ml of 50 mM Tris-HCl (pH 7.4) containing150 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 wasrepeated twice. The resulting pellet was washed five times with 100 mMsodium acetate (pH 5.5) containing 1 mM EDTA and treated with a mix-ture 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) contain-ing 150 mM NaCl at 37°C for 2 h. The supernatant (100 �l) obtained aftercentrifugation for 5 min at 22,000 � g at 4°C was subjected to cold acetoneprecipitation. The precipitate obtained after centrifugation at 22,000 � gfor 10 min was dissolved in 10 �l of SDS-PAGE sample buffer. The super-natant after the centrifugation of the homogenized cells described above

was centrifuged at 22,000 � g for 15 min, and the pellet was collected as amembrane fraction.

The membrane fraction was washed with buffer A three times, sus-pended in 300 �l of buffer A containing 1% (vol/vol) Triton X-100, andheld for 1 h at 4°C. The supernatant obtained by centrifugation at 22,000 � gfor 15 min was used as a membrane-solubilized fraction. The membrane-solubilized fraction (100 �l) was subjected to cold-acetone precipitation.The pellet obtained by centrifugation at 22,000 � g for 5 min was air driedand dissolved in 20 �l of SDS-PAGE sample buffer before immunoblotanalysis.

Phosphatidylinositol-specific phospholipase C treatment of themembrane fraction. The membrane fraction was incubated in 100 �l of50 mM Tris-HCl (pH 7.5) containing 5 mM EDTA and phosphatidyli-nositol-specific phospholipase C (PI-PLC) (0.4 U; Sigma-Aldrich) at 30°Cfor 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 10min, the supernatant contained the PI-PLC-soluble fraction. The pelletwas 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 supernatantobtained after the centrifugation of this solution at 22,000 � g for 10 minwas precipitated with acetone, and the pellet was used as the PI-PLC-insoluble fraction for immunoblot analyses.

Immunological analysis. Proteins were subjected to SDS-PAGE fol-lowed by blotting onto a membrane. The membrane was preincubated inPBST (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.1% [vol/vol] Tween20) containing 1.5% (wt/vol) nonfat milk for 1 h at room temperature andthen incubated for 1 h with a horseradish peroxidase-conjugated mono-clonal antibody against the polyhistidine tag (Qiagen) or the FLAG tag(Sigma-Aldrich), diluted 1:10,000 in PBST. After the membrane waswashed four times with PBST for 10 min, the antibody-antigen complexwas detected by using an ECL advanced detection kit (GE Healthcare) andan LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan).

Effect of Lam16A on glucose production from laminarin byUeBgl3A. Reaction mixtures (20 �l) containing Lam16A (0 to 2.0 �g),recombinant U. esculenta �-glucosidase (UeBgl3A) (0.02 �g) producedby A. oryzae (31), 0.1% (wt/vol) laminarin, and 100 mM sodium phos-phate (pH 6.0) were incubated at 30°C for 30 min. The amount of glucosewas determined by using a glucose oxidase assay kit (Megazyme) and wascalculated 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 hypertrophicstages of 1, 20, and 170 g of fresh weight) and first-strand cDNA synthesiswere carried out as described above. Reverse transcription (RT)-PCR wasperformed by using primers 5=-ACTCGTCGCCATGGAACGATCTTTCGG-3= and 5=-GTTTGCGGTGCTACCACCAATAGTGTAG-3=. ActinDNA was amplified by PCR using primers 5=-GACGGACAGGTGATCACCATTGGCAAC-3= and 5=-CTCCTGCTTCGAGATCCACATCTGCTG-3= to standardize reaction conditions.

Nucleotide sequence accession number. The sequence of the geneencoding Lam16A has been deposited in the DNA Data Bank of Japan(DDBJ) under accession number AB691944.

RESULTSPurification of laminarin-degrading enzyme. In a previousstudy, we reported that U. esculenta secreted enzymes responsiblefor laminarin degradation in culture medium containing glucoseas the sole carbon source (32). In the present study, we investi-gated a protein from a U. esculenta culture filtrate with endotypichydrolyzing activity on laminarin. When the culture filtrate wasloaded onto an anion-exchange column, laminarin-degrading ac-tivity was detected in the unbound fraction. The application of theunbound fraction onto a Toyopearl CM-650 column, a weak-cation-exchange column, also resulted in laminarin-degrading ac-tivity eluting in the unbound fraction (Fig. 1A). When the major

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enzymatically active fraction (fraction 2) (Fig. 1A) was loadedonto an UNOsphere S column, a strong-cation-exchange column,the major activity was detected in fractions 11 to 13, in which threemajor proteins of 60, 40, and 30 kDa were observed by silver stain-ing (Fig. 1B).

Identification of laminarin-degrading enzyme. The threemajor proteins were subjected to chymotrypsin digestion and an-alyzed by liquid chromatography-tandem mass spectrometry(LC/MS/MS) (see the supplemental material). Three peptide se-quences from the 60-kDa protein, two of which contained onemismatch each, were identical to those found in a putative GH16�-1,3-glucanase from S. reilianum SRZ2 (Table 1). Two peptidesequences were obtained from the 40-kDa protein, one of which isannotated as a phosphatidylethanolamine-binding protein. Theother showed no conserved domains based on a domain search atthe NCBI website. One peptide sequence obtained from the 30-kDa protein is not annotated as any conserved domain. Thus, itwas concluded that the 60-kDa protein was responsible for lami-narin degradation.

Sequence analysis. The gene encoding the 60-kDa protein, theU. esculenta laminarin-degrading enzyme belonging to GH16(Lam16A), was amplified by PCR from a U. esculenta cDNA poolusing degenerate primers based on the S. reilianum SRZ2 GH16�-glucanase gene. The cloned DNA consisted of 1,196 bp, includ-ing a predicted open reading frame of 1,173 bp. The translatedamino acid sequence indicates that Lam16A contains the con-served GH16 catalytic domain, a secretion signal peptide consist-ing of 25 amino acids at the N terminus, and a GPI anchor signalpeptide consisting of 29 amino acids at the C terminus. The N-ter-minal isoleucine in the amino acid sequence NRAGGGIIAMERSFfrom S. reilianum SRZ2 is replaced by leucine in Lam16A. Thismismatch occurs because isoleucine and leucine have the samemolecular weights. A phylogenetic tree of GH16 proteins showsthat Lam16A belongs to the Basidiomycetes group, with a clearseparation from Crh family proteins and characterized �-1,3-glu-canases, largely from eukaryotes (see Fig. S1 in the supplementalmaterial). Among Lam16A homologs, the GPI anchor signal pep-

tide is found only in proteins from the Ustilaginomycotina sub-phylum.

Enzymatic properties of recombinant Lam16A-His7. In or-der to generate recombinant Lam16A, 5 C-terminal amino acidresidues from Lam16A were replaced by heptahistidine residues(His7), because native Lam16A fused with C-terminal His7 couldnot be achieved (data not shown). Recombinant Lam16A-His7

overexpressed in A. oryzae was purified by using polyhistidineaffinity chromatography before enzymatic properties were deter-mined. Purified Lam16A-His7 exhibited a single broad band ataround 110 kDa by Sypro ruby staining (see Fig. S2A in the sup-plemental material). Immunoblot analysis using an antibodyagainst polyhistidine showed a single band with a molecular massidentical to that observed by Sypro ruby staining (see Fig. S2B inthe supplemental material). The enzyme was detected clearly byglycoprotein analysis (see Fig. S2C in the supplemental material),indicating that recombinant Lam16A-His7 was highly glycosy-lated, because Lam16A-His7 has a calculated molecular mass of 39kDa, with 7 potential N-glycosylation sites and 9 potential O-gly-cosylation sites.

The maximum hydrolytic activity on laminarin was observedat pH 5.0 and at 40°C (Fig. 2A and B). The enzyme retained over80% residual activity after incubation at 10°C to 40°C or pH 3.5 to7.0 for 1 h (Fig. 2C and D).

Substrate specificity. To investigate substrate specificity, thehydrolytic activity toward polysaccharides was determined;among the polysaccharides tested, Lam16A-His7 exhibited activ-ity only on �-1,3-glucan (Table 2), indicating that the enzyme ishighly specific for �-1,3-glucan hydrolysis. The activity towardlaminarioligosaccharides showed that Lam16A-His7 preferen-tially hydrolyzed laminarioligosaccharides with a degree of po-lymerization of 4 (Table 2). Lam16A exhibited the highest ac-tivity toward laminariheptaose among the substrates tested,showing a Km of 0.65 0.21 mM and a kcat of 21 2.2 s�1. Theend products generated from the hydrolysis of laminarioligosac-charides were laminaribiose and laminaritriose, and transglycosy-lation activity toward laminarioligosaccharides was not observed(data not shown).

Effect of the GPI anchor signal peptide on localization ofLam16A-FLAG. The Lam16A localization in an A. oryzae trans-formant overexpressing Lam16A-FLAG or Lam16A�GPI-FLAGwas determined immunologically in order to investigate the roleof the putative GPI anchor signal peptide. Lam16A-FLAG wasfound in the membrane and extracellular fractions but not in thecell wall fraction, whereas Lam16A�GPI-FLAG was detected onlyin the extracellular fraction (Fig. 3A). Similarly, higher hydrolyticactivity toward laminarin was detected in the membrane fraction

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

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

Peptide sequencea Species

DDBJaccessionno. Annotation

GTTGKGVRVW S. reilianumSRZ2

CBQ72681.1 GH16�-glucanase

NRAGGGIIAMERSF S. reilianumSRZ2

CBQ72681.1 GH16�-glucanase

NQSGCNAQYPACSY S. reilianumSRZ2

CBQ72681.1 GH16�-glucanase

a Underlining indicates a mismatched amino acid.

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from the transformant overexpressing Lam16A-FLAG and in theextracellular fraction from the transformant overexpressingLam16A�GPI-FLAG (Fig. 3B and C). The results from the en-zyme assay are consistent with those from the immunoblot anal-ysis. These results indicate that the GPI anchor signal peptide playsa significant role in the localization of Lam16A in the membrane.

Effect of PI-PLC treatment on localization of Lam16A-FLAG. The effect of PI-PLC treatment on the localization ofLam16A-FLAG was examined. PI-PLC treatment resulted in

Lam16A-FLAG localizing in the PI-PLC-soluble fraction, whereasthe enzyme remained in the membrane fraction after treatmentwith heat-inactivated PI-PLC (Fig. 4). Immunoblot analysis re-vealed two and three bands representing Lam16A-FLAG, likelydue to a variable degree of glycosylation. These results imply thatthe digestion of the GPI anchor with PI-PLC released Lam16Afrom the membrane.

Enhanced glucose production by the action of Lam16A. Glu-cose production from laminarin using purified Lam16A and

FIG 2 Effect of temperature and pH on hydrolytic activity of recombinant Lam16A-His7. The hydrolytic activity of Lam16A toward laminarin was assayed undervarious conditions. (A) The optimal temperature was determined after reaction mixtures containing laminarin, sodium acetate buffer (100 mM; pH 5.0), andLam16A (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 wasdetermined 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 5.0to 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.

TABLE 2 Substrate specificity of Lam16Aa

SubstrateMean sp act(U/mg) SE

Relativeactivity (%)

Laminarin, 0.02% 6.2 0.28 100Laminaribiose NDLaminaritriose NDLaminaritetraose 0.95 0.11 15Laminaripentaose 5.7 0.56 92Laminarihexaose 12 0.27 190Laminariheptaose 16 2.4 260CM-curdlan, 0.02% 10 0.083 165CM-pachyman, 0.02% 4.1 0.17 66a ND indicates that the activity was less than 0.1 U/mg of specific activity. The specificactivity of Lam16A toward lichenan, barley �-glucan, CM-cellulose, HE-cellulose,tamarind xyloglucan, arabinan, arabinogalactan, polygalacturonate, xylan, orpachyman as the substrate was less than 0.1 U/mg.

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) Thehydrolytic activity of the membrane (B) and extracellular (C) fractions wasdetermined by using laminarin as a substrate.

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UeBgl3A was assayed (Fig. 5). UeBgl3A produced 1.78 �g glucosefrom laminarin in this experiment, whereas Lam16A (2.0 �g) re-leased only 0.64 �g glucose. The levels of glucose production wereincreased as the amount of added Lam16A increased. Maximalglucose production (6.94 �g) was attained in the presence of 1.0�g Lam16A, about 4-fold higher than that produced by UeBgl3Aonly. This result suggests that the production of laminarioligosac-charides from laminarin by the action of Lam16A enhanced glu-cose production by UeBgl3A.

The expression levels of lam16A and UeBgl3A were examined byPCR to investigate their involvement during Z. latifolia gall forma-tion. High levels of lam16A transcripts were detected at the initialstage of gall formation (Fig. 6), and UeBgl3A was expressed constitu-tively. This result may suggest that Lam16A is involved in cell wallloosening, 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 in-volved in laminarin degradation. Lam16A was able to degrade�-1,3-glucan specifically and preferred substrates with a degree ofpolymerization of 4. UeBgl3A, a �-glucosidase from U. escu-lenta, produces glucose from a variety of �-glucosides. Laminari-oligosaccharides with degrees of polymerization of 2 to 4 havebeen reported to be substrates that are easily hydrolyzed by thisenzyme (32). The cooperative activity of Lam16A and UeBgl3A issuggested to be involved in the digestion of �-1,3-glucan to glu-cose efficiently during the growth of U. esculenta in culture me-dium and in Z. latifolia (Fig. 5 and 6).

Lam16A was partially purified from the culture filtrate, indi-cating that the enzyme is secreted extracellularly. However, aminoacid sequence analysis of Lam16A revealed a C-terminal GPI an-chor signal peptide that indicates a plasma membrane location.Recent proteomic analyses have shown that many proteins pos-sessing GPI anchor signal peptides from Aspergillus nidulans andS. cerevisiae are covalently linked to the cell wall (6, 12, 14, 15, 17,48), suggesting a possible cell wall location for Lam16A. Whilecomputational predictions would be helpful, to our knowledge,systematic sequence-based predictions of cell wall localizationhave not yet been performed for proteins in any organism. Toinvestigate the function of a possible GPI anchor signal peptide inLam16A, the localization of recombinant Lam16A-FLAG andLam16A�GPI-FLAG in A. oryzae was detected immunologically.Most of the Lam16A-FLAG protein was found in the membraneand extracellular fractions (Fig. 3A). Conversely, Lam16A�GPI-FLAG, lacking the GPI anchor signal peptide, was found only inthe extracellular fraction. Furthermore, the treatment of themembrane fraction from A. oryzae with PI-PLC released Lam16A-FLAG into the PI-PLC-soluble fraction (Fig. 4). These results ledto the conclusion that the GPI anchor signal peptide in Lam16Aplays a significant role in its membrane localization with the GPIanchor. GPI-anchored proteins in A. fumigatus have been re-ported to be released from membrane preparations by endoge-nous PI-PLC (4). Similarly, Lam16A found in extracellular frac-tions from U. esculenta and A. oryzae might be released by theaction of endogenous PI-PLC. The proteolytic release of Lam16Amight also occur by analogy to yeast Crh2p, which is released fromthe membrane depending on the activity of the transmembraneprotease Yps1p (30). The hydrolytic activity of the membranefraction from A. oryzae overexpressing Lam16A-FLAG was muchhigher than that of the membrane fraction from theLam16A�GPI-FLAG-overexpressing strain (Fig. 3B), indicatingthat Lam16A in the membrane is enzymatically active. Based onthese results, we propose that Lam16A is transferred to the plasmamembrane, where the enzyme hydrolyzes �-1,3-glucan, and thatthe enzyme is used even after it is released extracellularly.

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

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

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. Lane1, stage before hypertrophy; lanes 2, 3, and 4, 1 g, 20 g, and 170 g gall freshweight of hypertrophic stage, respectively. Bars represent 5 cm. (B) Transcriptlevels of lam16A and UeBgl3A were analyzed by RT-PCR. Amplified DNAfragments were stained with ethidium bromide.

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Lam16A-His7 was found to hydrolyze �-1,3-glucan specificallyand to act most efficiently on substrates with a degree of poly-merization of 4 (Table 2). This suggests cooperative activity withUeBgl3A, which acts preferentially on laminarioligosaccharideswith a degree of polymerization of 2 to 4 to produce glucose.Lam16A did not exhibit transglycosylation activity toward lami-narioligosaccharides, unlike Eng2, possessing a GPI anchor signalpeptide, a GH16 endo-�-1,3(4)-glucanase with transglycosylationactivity toward laminaritetraose (18), or Crh family proteins (7).Such a range of activity may have evolved by a process of accumu-lated DNA substitutions, as seen in the diversity of amino acidsequences in the GH16 phylogenetic tree (see Fig. S1 in the sup-plemental material), in which the GPI anchor signal is conservedonly in the Ustilaginomycotina subphylum among the Basidiomy-cetes cluster.

Together, these results suggest that GPI-anchored Lam16Amodifies the inner surface of the endogenous cell wall and after-wards is secreted to cleave �-1,3-glucan randomly to loosen thecell wall of the pathogen and/or the host, allowing for the catabolicuse of �-1,3-glucan through the cooperative action of �-glucosi-dases. This possibility is supported by previously reported obser-vations that Utr2, Crh11, and Crh12, all GPI-anchored cell wallproteins, play significant roles in cell wall formation (6, 34), andpea cellulase localizes at the inner surface of cell wall in auxin-treated pea epicotyl (2). Furthermore, as �-1,3-glucan and its sul-fated derivative form become elicitor molecules that induce de-fense responses in various plants, their catabolism would play animportant additional role in depressing the activation of host de-fense mechanisms (1, 20, 21, 28). During gall formation, Lam16Acould function to modify the U. esculenta cell wall and to degradeplant �-1,3-glucan (callose) in the host cell wall.

The synthesis and degradation of callose are involved in plantgrowth and development, plasmodesma regulation, and the stressresponse (11), even though callose is only a minor cell wall com-ponent. The action of Lam16A may also cause morphologicalchanges, especially during gall formation in Z. latifolia, whenlam16A is highly expressed. We anticipate that the findings re-ported here on the localization and hydrolytic activity of a novelGPI-anchored �-1,3-glucanase will lead to a greater understand-ing of the significance of cell wall modifications by hydrolytic en-zymes during hyphal extension and fungal growth and their ef-fects on host plant morphogenesis.

ACKNOWLEDGMENTS

We thank R. Oba, M. Kikuchi, and M. Iwai for technical assistance inpreparing plasmid DNA and transforming A. oryzae.

This work was supported in part by grant no. 23780111 from theJapanese Society for the Promotion of Science.

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