This article was downloaded by: [Adams State University]On: 18 November 2014, At: 09:09Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Bioscience, Biotechnology, and BiochemistryPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tbbb20

Characterization of Endo-β-N-acetylglucosaminidasefrom Alkaliphilic Bacillus halodurans C-125Kiyotaka FUJITAa, Hideto TAKAMIb, Kenji YAMAMOTOa & Kaoru TAKEGAWAc

a Division of Integrated Life Science, Graduate School of Biostudies, Kyoto Universityb Japan Marine Science and Technology Center, Microbial Genome Research Groupc Department of Life Sciences, Faculty of Agriculture, Kagawa UniversityPublished online: 22 May 2014.

To cite this article: Kiyotaka FUJITA, Hideto TAKAMI, Kenji YAMAMOTO & Kaoru TAKEGAWA (2004) Characterization ofEndo-β-N-acetylglucosaminidase from Alkaliphilic Bacillus halodurans C-125, Bioscience, Biotechnology, and Biochemistry,68:5, 1059-1066, DOI: 10.1271/bbb.68.1059

To link to this article: http://dx.doi.org/10.1271/bbb.68.1059

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Characterization of Endo-�-N-acetylglucosaminidase from

Alkaliphilic Bacillus halodurans C-125

Kiyotaka FUJITA,1 Hideto TAKAMI,2 Kenji YAMAMOTO,1 and Kaoru TAKEGAWA3;y

1Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan2Japan Marine Science and Technology Center, Microbial Genome Research Group,

2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan3Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received October 27, 2003; Accepted January 26, 2004

The genome sequencing project on alkaliphilic Bacil-

lus halodurans C-125 revealed a putative endo-�-N-

acetylglucosaminidase (Endo-BH), which consists of a

signal peptide of 24 amino acids, a catalytic region of

634 amino acids exhibiting 50.1% identity with the

endo-�-N-acetylglucosaminidase from Arthrobacter pro-

tophormiae (Endo-A), and a C-terminal tail of 220

amino acids. Transformed Escherichia coli cells carry-

ing the Endo-BH gene exhibited endo-�-N-acetylgluco-

saminidase activity. Recombinant Endo-BH hydrolyzed

high-mannose type oligosaccharides and hybrid type

oligosaccharides, and showed transglycosylation activ-

ity. On deletion of 219 C-terminal amino acid residues

of Endo-BH, the wild type level of activity was retained,

whereas with deletions of the Endo-A homolog domain,

the proteins were expressed as inclusion bodies and

these activities were reduced. These results suggest that

the enzymatic properties of Endo-BH are similar to

those of Endo-A, and that the C-terminal tail does not

affect the enzyme activity. Although the C-terminal tail

region is not essential for enzyme activity, the sequence

is also conserved among endo-�-N-acetylglucosamini-

dases of various origins.

Key words: endo-�-N-acetylglucosaminidase; Bacillus

halodurans; transglycosylation

Endo-�-N-acetylglucosaminidase (EC 3.2.1.96) cata-lyzes the hydrolysis of the N,N0-diacetylchitobiosemoiety of asparagine-linked oligosaccharides of variousglycoproteins. This enzyme from several differentsources such as animals, plants, fungi, and bacteria hasbeen characterized. Endo-�-N-acetylglucosaminidase isclassified into two glycoside hydrolase (GH) families, 18and 85, based on amino acid sequence similarities.1) Wepreviously cloned endo-�-N-acetylglucosaminidasefrom Arthrobacter protophormiae (Endo-A), which isclassified into GH family 85.2) Endo-A has been used forthe synthesis of neoglycoconjugates and neoglycopro-

teins due to its strong transglycosylation activity towardhigh-mannose type oligosaccharides.3–7) We identifiedE173 as a catalytically essential amino acid and W216as a critical amino acid for transglycosylation by PCRrandom and site-directed mutagenesis.8) GH family 85homologs were recently cloned from Mucor hiemalis(Endo-M),9) Streptococcus pneumoniae (Endo-D),10)

Caenorhabditis elegans (Endo-CE),1) and Homo sapi-ens.11) The essential amino acids are conserved in thesehomologs (except for Endo-D), but the catalytic mech-anism and substrate recognition characteristics are notwell understood. Endo-M and Endo-CE can act oncomplex-type oligosaccharides,10,12–15) whereas Endo-Ais limited to high-mannose and hybrid-type oligosac-charides. But Endo-A is the only well characterizedbacterial endo-�-N-acetylglucosaminidase that exhibitstransglycosylation activity. To elucidate the reason forthe difference in substrate recognition, we have attempt-ed to find other GH family 85 enzymes. Genomicsequencing projects have revealed the presence ofputative proteins exhibiting sequence similarity to GHfamily 85 enzymes from bacteria, fungi, insects, plants,and mammals.An alkaliphilic bacterium, Bacillus halodurans strain

C-125, grows well in alkaline environments (pH range,6.8 to 10.8), and produces many alkaline enzymes, suchas proteases, amylases, and cellulases.16) These alkalineenzymes are widely used as industrial products. Thecomplete genome sequence of B. halodurans has beendetermined and contains 4,066 predicted protein codingsequences.17) We inspected the current genome databasefor B. halodurans, and it revealed the presence of oneputative endo-�-N-acetylglucosaminidase (Endo-BH)gene (GenBank: AP001509) exhibiting 50% identitywith Endo-A in 634 amino acids of the N-terminalregion. In this study, we describe the functionalcharacterization of the gene encoding a homolog ofendo-�-N-acetylglucosaminidase.

y To whom correspondence should be addressed. Tel: +81-87-891-3116; Fax: +81-87-891-3021; E-mail: takegawa@ag.kagawa-u.ac.jp

Abbreviations: Man, mannose; GlcNAc, N-acetylglucosamine; Dns-GP, dansylated glycopeptide; SGP, sialylglycopeptide

Biosci. Biotechnol. Biochem., 68 (5), 1059–1066, 2004

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

Materials and Methods

Bacterial strains, plasmids, and media. AlkaliphilicB. halodurans C-125 was cultivated in Horikoshi IImedium, containing 1% soluble starch, 0.5% tryptone,0.5% yeast extract, 0.1% K2HPO4, 0.1% MgSO4�7H2O,and 2.0% NaHCO3 (separately autoclaved), pH 9.5.Escherichia coli XL1-Blue (Stratagene, La Jolla, CA)was used for plasmid construction and as the host forcloning vector pCR 2.1 (Invitrogen, Carlsbad, Califor-nia). E. coli BL21 (DE3) was used as the host forplasmid pET-23d (Novagen, Madison, Wisconsin). AllE. coli strains were cultivated in Luria–Bertani medium(LB).

Preparation of cell extracts for Western immunoblot-ting. SDS extracts were made from B. haloduranscultures to OD600 of 1.0 in potassium phosphate buffer(pH 7.0) as follows. The cells were harvested bycentrifugation, washed with 10mM potassium phosphatebuffer (pH 7.0), resuspended in 80�l of SDS samplebuffer (125mM Tris–HCl buffer [pH 6.8], 2% SDS, 5%2-mercaptoethanol, 0.002% bromophenol blue, 5%Sucrose), and heated at 100 �C for 5min. The super-natant was analyzed by Western immunoblotting.

Enzyme assay and analysis of transglycosylationproducts. Endo-�-N-acetylglucosaminidase activitywas assayed with dansylated Man6GlcNAc2Asn as thesubstrate. One unit was defined as the amount yielding1�mol of GlcNAc–Asn–dansyl per minute at 37 �Cunder the assay conditions described preciously.18)

Man6GlcNAc2Asn was prepared from ovalbumin gly-copeptides by the method of Huang et al.19)

The transglycosylation reaction was performed asfollows: 1.3mg of Man6GlcNAc2Asn, as the glycosidedonor, was incubated with 10 munits of the enzyme in0.27ml of 50mM ammonium acetate buffer (pH 6.0) inthe presence of each glycoside acceptor (final concen-tration, 0.1 M). After incubation of the mixture for10min at 37 �C, the reaction was stopped by boiling for3min. The reaction mixture was concentrated bylyophilization and then subjected to thin-layer chroma-tography (TLC) on a silica gel 60 plate (Merck Art.5626). For the separation and purification of theoligosaccharides, the following solvent system wasused: n-propanol/acetic acid/water ðv=vÞ ¼ 3=3=2. Or-cinol–H2SO4 reagent was used for detection of theoligosaccharides.20) The transglycosylation productsdetected on TLC plates were quantitated by means ofNIH-Image software (version 1.63).

Construction of expression plasmids and their ex-pression. The Endo-BH gene was amplified from thegenome fragment of B. halodurans C-125 CC0516s/CC0516a using an upstream primer (300-bp upstream ofthe start codon), 50-ATTAAAGGGATTGGCAAGGC-30, and a downstream primer (124-bp downstream of the

stop codon), 50-GACGCCTACACTAGTATTGTC-30.The amplified fragments were inserted into the pCR2.1 vector, the fragment being inserted in the reverseorientation to the inducible lac promoter. The construct-ed plasmid was then transformed into E. coli XL1-Blue.

The coding region of the putative endo-�-N-acetyl-glucosaminidase gene was amplified from the pCR 2.1-derived clone by PCR using the F�15 primer, 50-GTCAAGCCATGGCAAGTCAGCCTGAG-30, as theforward primer, and a C-terminal primer, 50-CCT-GGATCCGATAAA-TTCGTGCCGC-30, as the reverseprimer. The forward primer was designed to delete theputative signal peptide. The restriction sites used forsubcloning of the PCR products are given in italicletters. The amplified fragment was digested with NcoIand BamHI, and then inserted into the NcoI and BamHIsites of the pET-23d (+) vector. The plasmid carryingthe Endo-BH gene with a C-terminal His–Tag wastransformed into E. coli BL21 (DE3). E. coli cellscontaining the plasmid were grown in LB containing100�g/ml ampicillin at 37 �C until the absorbance at600 nm reached 0.4, and then induced by adding 0.4mM

(final concentration) IPTG at 37 �C for 3 h. The cellswere collected by centrifugation and the cell pellet wasresuspended in BugBuster protein extraction reagent(Novagen). The soluble extract was purified withHisTrap Kit metal chelation resin (Amersham Bio-sciences, Uppsala, Sweden).

Partial purification of Endo-BH from B. halodurans.Endo-BH from B. halodurans was partially purifiedfrom 100ml of culture broth after cultivation on a shakerat 37 �C for 55 h. After centrifugation, ammoniumsulfate was added to the supernatant to 80% saturation.The precipitated protein was collected by centrifugation,dissolved in 10mM potassium phosphate buffer(pH 7.0), dialyzed against the same buffer, and thenapplied to a DEAE-Sepharose FF column (6� 20 cm;Amersham Biosciences) equilibrated with the samebuffer. The enzyme was eluted with 0.4 M NaCl in thesame buffer. The active fractions were concentratedwith an Ultrafree 10K NMWL membrane (Millipore,Bedford, Massachusetts).

Mutageneses. The expression plasmids for deletionmutants of Endo-BH were constructed by PCR using theF�15 primer and one of five oligonucleotides as thereverse primer: R�249, 50-AGCGTTGGATCCTCATT-CCTTCCCAACTC-30; R�239, 50-AAACGTGGATCC-TCAAACAGGAACCAC-30; R�229, 50-AAACGTG-GATCCTCAACCAGATGGACGAC-30; R�219, 50-TCTCGGGGATCCATATGGTGGCCACTC-30; andR�199, 50-ATTATTGGATCCTCAAATCGACTCAC-CTGG-30 (italics indicate a BamHI site).

Site-directed mutagenesis was performed withpET23d plasmid carrying the Endo-BH gene as atemplate according to the procedure for a QuikChangesite-directed mutagenesis kit (Stratagene). Mutations

1060 K. FUJITA et al.

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

were confirmed by DNA sequencing. The plasmidscontaining mutated Endo-BH were transformed intoE. coli BL21 (DE3). The transformants were inducedwith 0.4mM IPTG for 3 h at 37 �C. The mutants wereextracted with BugBuster protein extraction reagent andthen purified on a HisTrap Kit. Two primers, 50-GG-TTTATTAAtCAAcAAACAGAAGGAGGG-30 and 50-CCTTCTGTTTgTTGaTTAATAAACCAACC-30, wereused to generate the E172Q mutation. And two primers,50-GGAGACATTCGCcGGCAAAACTATTT-30 and 50-AAATAGTTTTGCCgGCGAATGTCTCC-30, wereused to generate the W215R mutation. The mutatedsequences are shown in lowercase letters.

Antibody preparation and immunoblotting. Anti-Endo-BH antiserum was prepared as follows. Purifiedrecombinant Endo-BH protein was subjected to 10%SDS-PAGE. The gel was stained with Coomassie blue.The electroeluted protein solution was dialyzed exhaus-tively against distilled water and then lyophilized.Approximately 1mg of the protein was emulsified withFreund’s complete adjuvant (Difco, Detroit, Michigan),and then injected into a young male New Zealand Whiterabbit. For Western blotting, proteins were subjected to10% SDS-PAGE and then electrotransferred onto poly-

vinylidene difluoride (PVDF) membranes. Antigen-antibody complexes were detected by reaction withperoxidase-conjugated anti-rabbit immunoglobulin G(Amersham Biosciences), and were developed with anECL detection kit (Amersham Biosciences).

Results

Presence of endo-�-N-acetylglucosaminidase activityin the culture broth of B. halodurans C-125We examined whether the culture broth of alkaliphilic

B. halodurans exhibits endo-�-N-acetylglucosaminidaseactivity. The culture broth of B. halodurans C-125 wasassayed for endo-�-N-acetylglucosaminidase activityafter cultivation for 3 days at 37 �C in Horikoshi IImedium at pH 9.5. Endo-�-N-acetylglucosaminidaseactivity was detected in the culture broth, and maximumactivity was observed after 55 h of cultivation (Fig. 1A).The expression of Endo-BH was also confirmed byWestern immunoblotting of the culture broth and SDS-treated cell extract (Fig. 1B). The amount of secretedenzyme increased depending on cell growth. The mo-lecular mass of the partially purified Endo-BH wasfound to be about 90 kDa (Fig. 2B, lane 1).

Fig. 1. Time-courses of Growth and Enzyme Production in a Culture of B. halodurans C-125.

The culture was carried out in a 500ml flask containing 100ml of medium. (A) Time-courses of growth and enzyme activities at pH 9.5. (B)

Western immunoblotting of various culture supernatants (6�l) (a) and SDS-treated cell extracts (10�l) (b) with anti-Endo-BH. Lane 1, 8 h;

lane 2, 11.5 h; lane 3, 14 h; lane 4, 16 h; lane 5, 26 h; lane 6, 40 h; lane 7, 55 h. The arrow indicates the position of Endo-BH produced by

B. halodurans.

Cloning of Bacillus Endo-�-N-acetylglucosaminidase 1061

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

Expression of Endo-BH in E. coli cells and itspurificationThe putative Endo-BH gene encodes a protein of 878

amino acids and consists of a predicted signal peptide of24 amino acids, a conserved region of 425 amino acidsexhibiting 65.4% identity with Endo-A, a narrowconserved region of 195 amino acids exhibiting 24.0%identity with Endo-A, and a non-conserved region of237 amino acids (Fig. 2A). The mature protein ispredicted to have a molecular mass of 96 kDa.The PCR amplified Endo-BH gene was subcloned

into the pCR 2.1 vector in the forward or reverseorientation and each plasmid was transformed intoE. coli XL1-Blue. In each case, enzyme activity wasdetected in the cell lysate. This result indicated that the

cloned Endo-BH gene was expressed by its ownpromoter. Next, the complete ORF of Endo-BH withoutthe predicted signal peptide (N�24) was inserted intoexpression plasmid pET23d. The recombinant enzymewas expressed in E. coli BL21 (DE3), and the solublecell lysate showed enzymatic activity. The His-taggedEndo-BH was purified from the cell lysate using aHisTrap kit. The molecular mass was estimated to beabout 96 kDa on SDS-PAGE (Fig. 2B, lane 2).

Enzymatic properties and transglycosylation activityof Endo-BH

Although B. halodurans C-125 produced variousenzymes (protease, xylanase, amylase, and pectinase)and these enzymes showed optimum pHs of around

Fig. 2. Alignment of GH Family 85 Enzymes and Expression of His-tagged Endo-BH in E. coli.

(A) The predicted cleavage sites of the signal peptide are indicated by arrows. The genes identified as endo-�-N-acetylglucosaminidase are

shown in bold characters after the name. The organisms and GenBank accession numbers are as follows: Bacillus halodurans C-125

(AP001509), Arthrobacter protophormiae AKU 0647 (U59168), Bifidobacterium longum NCC2705 (AE014762), Lactobacillus plantarum

WCFS1 (AL935252), Clostridium perfringens 13 (AP003186), Lactococcus lactis subsp. lactis IL1403 (AE006379), Streptococcus pneumoniae

TIGR4, Mucor hiemalis (AB060586), Pichia anomala (AJ306295), Anopheles gambiae str. (AAAB01008846), Drosophila melanogaster

(AY075262); Arabidopsis thaliana ORF At3g11040 (AC009991), ORF At5g05460 (AB010692), Homo sapiens (AF512564), Mus musculus

(AK048667), and Caenorhabditis elegans (AB079783). Completely conserved residues and conserved substitutions are highlighted in black and

dark gray respectively. Filled and open triangles indicate the residues corresponding to the important amino acids for the hydrolysis and

transglycosylation activity of Endo-A respectively. (B) (a) SDS-PAGE and (b) Western blot analysis of Endo-BH. Lane 1, partially purified

Endo-BH produced by B. halodurans; lane 2, purified recombinant Endo-BH; lane M, molecular weight markers. The samples were loaded onto

a 10% gel. Filled and open arrows indicate the positions of recombinant Endo-BH and B. halodurans-produced Endo-BH respectively.

1062 K. FUJITA et al.

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

pH 9–10,17) Endo-BH exhibited only 28% of maximumactivity at pH 10.0. The optimum pH of Endo-BH wasfound to be 6.0 to 7.0 at 37 �C, and above 60% activitywas retained at pH 9.0 (data not shown). The substratespecificity of the purified Endo-BH was examined usingdansylated asparaginyl oligosaccharides of ovalbumin.About 7 nmol of each of Dns-GP-I to -V was incubatedwith 0.64mU of the enzyme in 50mM acetate buffer(pH 6.0) at 37 �C for 5min (total volume, 10�l). Therelative activities of Endo-BH are shown in Table 1. Asa result, high-mannose oligosaccharides were found tobe suitable substrates for the enzyme, and the hydrolysisrate for Dns-GP-V was 66.0% of that for Dns-GP-IV.The results indicated that the existence of the Man�1–2Man structure in the branches of oligosaccharides isimportant for the enzymatic activity. The hydrolysisrates for hybrid type oligosaccharides were very low(Table 1). In addition, the enzyme did not act oncomplex-type oligosaccharide derived from SGP. Thesecharacteristics are similar to those of Endo-A,18) andEndo-BH appeared to act on high-mannose-type andhybrid-type oligosaccharides of glycoproteins. Theenzymatic properties of the native enzyme were com-pared with those of the recombinant enzyme, and bothenzymes showed similar subbstrate specificity (data notshown).

The transglycosylation of Man6GlcNAc2Asn to glu-cose, gentiobiose, and galactose by the enzyme wasexamined, as described under Materials and Methods.The transglycosylation products were identified as thecorresponding products produced by Endo-A in thepresence of glucose (67.5%) or gentiobiose (93.3%)

(Fig. 3, lanes a-1 and b-1), but could not be detected inthe presence of galactose (Fig. 3, lane c-1). Theseresults indicated that Endo-BH exhibited transglycosy-lation activity, and glucose and gentiobiose wereattached to the reducing-end GlcNAc during hydrolysis.These characteristics are similar to those of Endo-A.

Construction and characterization of Endo-BH mu-tantsIn the case of Endo-A, mutation of the Glu-173

Table 1. Relative Activity of Endo-BH toward Various DNS-Glycopeptides

DNS-glycopeptides

GP-V

GP-IV

GP-II

GP-I

SGP

Relativeactivity (%)

a

66.0

100

9.7

5.5

NDb

Structure

Manαl

ManαlManαl

63Manαl 6

3Man l 4GlcNAc l 4GlcNAc-Asn

Manαl

Manαl

Manαl 2Manαl

63Manαl 6

3Man l 4GlcNAc l 4GlcNAc-Asn

63Manαl

42Manαl

63Man l

[Man]αl

Manαl

GlcNAc l

44GlcNAc l 4GlcNAc-Asn

[Gal] l 4GlcNAc l

GlcNAc l

63Manαl

42Manαl

Manαl

Manαl

GlcNAc l

463 Man l 4GlcNAc l 4GlcNAc-Asn

Gal l 4GlcNAc l

GlcNAc l

Neu5Acα2

Neu5Acα2

6Gal l 4GlcNAc l 2Manαl

6Gal l 4GlcNAc l 2Manαl

63 Man l 4GlcNAc l 4GlcNAc-Peptide

a The value obtained with GP-IV was taken as 100.b ND, not detectable.

Fig. 3. TLC Analysis of the Transglycosylation Activity toward

Several Acceptors.

Man6GlcNAc2Asn was incubated with Endo-BH (lane 1) or

Endo-A (lane 2) in the presence of 0.1 M glucose (a), gentiobiose

(b), or galactose (c) at 37 �C for 10min. Lane D, standard

Man6GlcNAc2Asn; lane H, standard Man6GlcNAc. M, Man; GN,

GlcNAc.

Cloning of Bacillus Endo-�-N-acetylglucosaminidase 1063

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

residue to Gln completely abolished enzyme activity andthat of Trp-216 to Arg abolished transglycosylationactivity.7) These positions are commonly conservedamong GH family 85-type endo-�-N-acetylglucosami-nidases, as shown in Fig. 2A. To elucidate the roles ofthese amino acids in Endo-BH, mutants with Glu-172(corresponding to 173 in Endo-A) changed to Gly andGln, and Trp-215 (corresponding to 216 in Endo-A) toArg were constructed by site-directed mutagenesis. Themutant, E172Q, showed no activity (data not shown),and the W215R mutant showed lower transglycosylationreactivity than the wild-type enzyme (Fig. 4). Theseresults suggest that these amino acid residues areimportant for Endo-BH activity.We searched for conserved domains among the

bacterial GH family 85 enzymes by means of aHmmPfam search (Fig. 5A). Curiously, a region at theC-terminus of Endo-BH exhibited similarity to theputative endo-�-N-acetylglucosaminidases from Bifido-bacterium longum (Endo-BL), Lactobacillus plantarum(Endo-LP), and Clostridium perfringens (Endo-CL)(Fig. 5B). In order to determine whether or not the C-terminal region of Endo-BH is important for enzymeactivity, several deletion mutants were constructed, asshown in Fig. 5B. C�219 lacks 219 C-terminal residuesof Endo-BH, which is nearly the same size as the Endo-A protein. C�199, C�229, C�239, and C�249 lack199, 229, 239, and 249 C-terminal residues of Endo-BHrespectively. These deletion mutants were expressed inE. coli cells (Fig. 5C). The C�199 and C�219 mutantsappeared to be highly soluble as judged by SDS-PAGEanalysis (Fig. 5C(a)), whereas the C�229, C�239, andC�249 mutants were mostly expressed as inclusionbodies (Fig. 5C(b)). The specific activities of theaffinity-purified deletion mutants were compared withthose of the wild-type enzyme. The C�199, C�219,C�229, and C�239 mutants retained the specificactivity of the wild-type enzyme but the specific activityof the C�249 mutant was below 1%. In the case of

Endo-A, deletion of 20 C-terminal amino acid residuesreduced activity (data not shown). We confirmed thatdeletion of the C-terminal region does not affect thetransglycosylation of Endo-BH (data not shown). Theseresults indicate that the 634 amino acids of the Endo-Ahomologous region encode the activity of Endo-BH, andthat the 219 amino acids of the C-terminal region are notessential for the activity of Endo-BH.

Discussion

In this study, we confirmed that Endo-BH is thesecond endo-�-N-acetylglucosaminidase from bacteriathat exhibits transglycosylation activity. GH family 85enzymes include a (FY)(DEH)G(WY)(LF)(ILVF)NXEsegment comprising the amino acid residues at positions164 to 172 in Endo-BH, as shown in Fig. 2A. Theglutamic acid residue at position 172 is essential for theactivity of Endo-BH, and was predicted to be a commoncatalytic residue in GH family 85 enzymes on the basisof a computational analysis of GH families.21) Thetryptophan residue at position 215 in Endo-BH proved tobe important for transglycosylation activity, which wasfirst found for Endo-A, and this tryptophan residue ishighly conserved in almost all GH family 85 enzymes,the exception being Endo-D from S. pneumoniae.Therefore, Endo-BH might have the same mechanicaland structural characteristics as other GH family 85endo-�-N-acetylglucosaminidases.

We found that the C-terminal region of Endo-BHexhibits similarity to the putative endo-�-N-acetylglu-cosaminidases from B. longum, L. plantarum, andC. perfringens (Fig. 5B). A BLAST search revealedthat these sequences exhibit a weak homology tosequences of the cell surface proteins from Methano-sarcina acetivorans C2A (Q8THC9, Q8TPZ1, Q8TPY6,Q8TPY7, Q8TJS7, Q8TPZ7, Q8TI72) and Methano-bacterium thermoautotrophicum (O26812, F69195).One possible explanation is that the C-terminal regionof Endo-BH is essential for retention on the cell surfaceof bacteria. Actually, Endo-D containing an N-terminalsignal peptide and a C-terminal Gram-positive anchordomain was predicted to be a cell surface-associatedenzyme.10) But, Endo-BH, BL, and LP do not contain aC-terminal Gram-positive anchor, and we do not knowwhether the C-terminal region of Endo-BH contributesto the signal for localization to the cell surface ofB. halodurans. We have shown that the C-terminalregion is not essential for the hydrolytic or transglyco-sylation activity of Endo-BH, but on deletion of 229amino acids of Endo-BH, it was expressed as aninclusion body in E. coli (Fig. 5). This suggests thatthe C-terminal region prevents the aggregation of Endo-BH, and hence that these peptides may contribute to thestability or conformation of the Endo-BH protein. Weare currently examining the functional roles of the C-terminal region of Endo-BH by means of PCR randomand site-directed mutagenesis.

Fig. 4. Transglycosylation Activity of the Endo-BH W215R Mutant.

Man6GlcNAc2Asn was incubated with the wild-type Endo-BH

(A) or mutant W215R (B) in the presence of 0.1 M glucose at 37 �C

for 0min (lane 1), 10min (lane 2), 20min (lane 3), and 30min

(lane 4), as described in Materials and Methods. M, Man; GN,

GlcNAc.

1064 K. FUJITA et al.

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

Acknowledgments

This work was partly supported by a Grant-in-Aid forYoung Scientists from the Japan Society for thePromotion of Science (JSPS).

References

1) Kato, T., Fujita, K., Takeuchi, M., Kobayashi, K.,Natsuka, S., Ikura, K., Kumagai, H., and Yamamoto, K.,Identification of an endo-�-N-acetylglucosaminidasegene in Caenorhabditis elegans and its expression inEscherichia coli. Glycobiology, 12, 581–587 (2002).

2) Takegawa, K., Yamabe, K., Fujita, K., Tabuchi, M.,Mita, M., Izu, H., Watanabe, A., Asada, Y., Sano, M.,Kondo, A., Kato, I., and Iwahara, S., Cloning, sequenc-ing, and expression of Arthrobacter protophormiaeendo-�-N-acetylglucosaminidase in Escherichia coli.Arch. Biochem. Biophys., 338, 22–28 (1997).

3) Takegawa, K., Tabuchi, M., Yamaguchi, S., Kondo, A.,Kato, I., and Iwahara, S., Synthesis of neoglycoproteinsusing oligosaccharide-transfer activity with endo-�-N-acetylglucosaminidase. J. Biol. Chem., 270, 3094–3099(1995).

4) Fan, J. Q., Quesenberry, M. S., Takegawa, K., Iwahara,S., Kondo, A., Kato, I., and Lee, Y. C., Synthesis ofneoglycoconjugates by transglycosylation with Arthro-bacter protophormiae endo-�-N-acetylglucosaminidase.Demonstration of a macro-cluster effect for mannose-binding proteins. J. Biol. Chem., 270, 17730–17735(1995).

5) Takegawa, K., Fujita, K., Fan, J. Q., Tabuchi, M.,Tanaka, N., Kondo, A., Iwamoto, H., Kato, I., Lee, Y.C., and Iwahara, S., Enzymatic synthesis of a neo-glycoconjugate by transglycosylation with Arthrobacterendo-�-N-acetylglucosaminidase: a substrate for colori-metric detection of endo-�-N-acetylglucosaminidaseactivity. Anal. Biochem., 257, 218–223 (1998).

6) Yamamoto, K., Haneda, K., Iguchi, R., Inazu, T.,

Fig. 5. Schematic Drawings and Alignment of the C-Terminal Sequences of Endo-BH and Bacterial GH Family 85 Enzymes, and SDS-PAGE

Analysis of Deletion Mutants of Endo-BH.

(A) A conserved domain search was performed using HmmPfam. SP, signal peptide; anchor, Gram-positive anchor domain. (B) The regions of

alignment are shown in gray boxes. Completely conserved residues and conserved substitutions are highlighted in black and gray respectively.

The black bars indicate regions of deletion mutants of Endo-BH. (C) The deletion mutants of Endo-BH were extracted with BugBuster into the

supernatants of cell-free extracts (a) and insoluble inclusion bodies (b). Lane 1, wild type; lane 2, C�199; lane 3, C�219; lane 4, C�229;

lane 5, C�239; lane 6, C�249; lane M, molecular weight markers. The samples were loaded onto 10% gels. The gels were stained with

Coomassie Brilliant Blue. The arrows indicate the position of recombinant Endo-BH.

Cloning of Bacillus Endo-�-N-acetylglucosaminidase 1065

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014

Mizuno, M., Takegawa, K., Kondo, A., and Kato, I.,Chemo-enzymatic synthesis of a calcitonin derivativecontaining a high-mannose type oligosaccharide byendo-�-N-acetylglucosaminidase from Arthrobacterprotophormiae. J. Biosci. Bioeng., 87, 175–179 (1999).

7) Fujita, K., Tanaka, N., Sano, M., Kato, I., Asada, Y., andTakegawa, K., Synthesis of neoglycoenzymes withhomogeneous N-linked oligosaccharides using immobi-lized endo-�-N-acetylglucosaminidase A. Biochem. Bio-phys. Res. Commun., 267, 134–138 (2000).

8) Fujita, K., and Takegawa, K., Tryptophan-216 isessential for the transglycosylation activity of endo-�-N-acetylglucosaminidase A. Biochem. Biophys. Res.Commun., 283, 680–686 (2001).

9) Kobayashi, K., Takeuchi, M., Wakamatsu, A.,Yamamoto, K., Kumagai, H., and Yoshida, S., JapanKokai Tokkyo Koho 332568 (Dec. 7, 1999).

10) Muramatsu, H., Tachikui, H., Ushida, H., Song, X., Qiu,Y., Yamamoto, S., and Muramatsu, T., Molecularcloning and expression of endo-�-N-acetylglucosamini-dase D, which acts on the core structure of complex typeasparagine-linked oligosaccharides. J. Biochem., 129,923–928 (2001).

11) Suzuki, T., Yano, K., Sugimoto, S., Kitajima, K.,Lennarz, W. J., Inoue, S., Inoue, Y., and Emori, Y.,Endo-�-N-acetylglucosaminidase, an enzyme involvedin processing of free oligosaccharides in the cytosol.Proc. Natl. Acad. Sci. USA, 99, 9691–9696 (2002).

12) Yamamoto, K., Fujimori, K., Haneda, K., Mizuno, M.,Inazu, T., and Kumagai, H., Chemoenzymatic synthesisof a novel glycopeptide using a microbial endoglycosi-dase. Carbohydr. Res., 305, 415–422 (1998).

13) Haneda, K., Inazu, T., Mizuno, M., Iguchi, R., Tanabe,H., Fujimori, K., Yamamoto, K., Kumagai, H., Tsumori,K., and Munekata, E., Chemo-enzymatic synthesis of abioactive peptide containing a glutamine-linked oligo-saccharide and its characterization. Biochim. Biophys.Acta, 1526, 242–248 (2001).

14) Haneda, K., Inazu, T., Mizuno, M., Iguchi, R.,Yamamoto, K., Kumagai, H., Aimoto, S., Suzuki, H.,and Noda, T., Chemo-enzymatic synthesis of calcitoninderivatives containing N-linked oligosaccharides.Bioorg. Med. Chem. Lett., 8, 1303–1306 (1998).

15) Saskiawan, I., Mizuno, M., Inazu, T., Haneda, K.,Harashima, S., Kumagai, H., and Yamamoto, K.,Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysisof its biological activity. Arch. Biochem. Biophys., 406,127–134 (2002).

16) Takami, H., and Horikoshi, K., Analysis of the genomeof an alkaliphilic Bacillus strain from an industrial pointof view. Extremophiles, 4, 99–108 (2000).

17) Takami, H., Nakasone, K., Takaki, Y., Maeno, G.,Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura,Y., Ogasawara, N., Kuhara, S., and Horikoshi, K.,Complete genome sequence of the alkaliphilic bacteriumBacillus halodurans and genomic sequence comparisonwith Bacillus subtilis. Nucleic Acids Res., 28, 4317–4331(2000).

18) Takegawa, K., Fujiwara, K., Iwahara, S., Yamamoto, K.,and Tochikura, T., Induction and purification of endo-�-N-acetylglucosaminidase from Arthrobacter protophor-miae grown in ovalbumin. Appl. Environ. Microbiol., 55,3107–3112 (1989).

19) Huang, C. C., Mayer, H. E. J., and Montgomery, R.,Microheterogeneity and paucidispersity of glycopro-teins; Part 1. The carbohydrate of chicken ovalbumin.Carbohydr. Res., 13, 127–137 (1970).

20) Holmes, E. W., and O’Brien, J. S., Separation ofglycoprotein-derived oligosaccharides by thin-layerchromatography. Anal. Biochem., 93, 167–170 (1979).

21) Rigden, D. J., Jedrzejas, M. J., and de Mello, L. V.,Identification and analysis of catalytic TIM barreldomains in seven further glycoside hydrolase families.FEBS Lett., 544, 103–111 (2003).

1066 K. FUJITA et al.

Dow

nloa

ded

by [

Ada

ms

Stat

e U

nive

rsity

] at

09:

09 1

8 N

ovem

ber

2014