Carbohydrate Research 375 (2013) 29–34

Contents lists available at SciVerse ScienceDirect

Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Note

TMG-chitotriomycin as a probe for the prediction of substratespecificity of b-N-acetylhexosaminidases

0008-6215/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carres.2013.04.024

⇑ Corresponding author. Tel.: +81 86 251 8291; fax: +81 86 251 8388.E-mail address: nitoda@cc.okayama-u.ac.jp (T. Nitoda).

Hiroto Shiota a, Hiroshi Kanzaki a, Tadashi Hatanaka b, Teruhiko Nitoda a,⇑a Laboratory of Bioresources Chemistry, The Graduate School of Environmental & Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japanb Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries, Research Institute for Biological Sciences, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 February 2013Received in revised form 5 April 2013Accepted 14 April 2013Available online 27 April 2013

Keywords:TMG-chitotriomycinb-N-AcetylhexosaminidaseGH20 familyInhibitorSubstrate specificityPhysiological function

TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was exam-ined as a probe for the prediction of substrate specificity of b-N-acetylhexosaminidases (HexNAcases).According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were dividedinto 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated fortheir substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides butnot N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligo-saccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as amolecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processingHexNAcases.

� 2013 Elsevier Ltd. All rights reserved.

b-N-Acetylhexosaminidase (HexNAcase, EC 3.2.1.52), a widelydistributed glycoside hydrolase, catalyzes the release of N-acetylh-exosamine from the non-reducing ends of substrates such as chitinoligomers and glycoconjugates. HexNAcase is responsible fornumerous physiological functions such as chitin degradation, gly-coconjugate processing, signal transduction, fertilization, seed ger-mination, and virus infection.1 Despite these various physiologicalroles, the basis for these functions has not been establishedcompletely.

Enzyme inhibitors are often used as powerful tools for thestructural and functional characterization of enzymes, which isalso the case for HexNAcase. Structures of HexNAcase inhibitors(1–3) used in this work are presented in Figure 1. Inhibitory spec-ificity of PUGNAc (2) and NAG-thiazoline (3) are reported to agreewith the classification of GH families (CAZy database at http://www.cazy.org/),2,3 namely, PUGNAc (2) inhibits all HexNAcasesincluding GH3, GH20, and GH84; NAG-thiazoline (3) specificallyinhibits GH20 and GH84 HexNAcases.4 However, a HexNAcaseinhibitor, TMG-chitotriomycin (1), which was isolated by ourgroup 5 from the culture filtrate of the actinomycete Streptomycesannulatus NBRC13369 and which was structurally revised byYoung et al.,6 was reported to have specific inhibition against Hex-NAcases from insects and fungi but no inhibition against thosefrom plants and mammals.5 Therefore, the inhibitory specificityof 1 was considered to correlate with the substrate specificity of

HexNAcases. Recently, Liu et al. reported on the structural basisfor the specialized function of HexNAcase from the Asian cornborer Ostrinia furnacalis (OfHex1) as a chitinolytic enzyme basedon the crystal structure of OfHex1 complexed with 1.7 They re-ported that all chitinolytic HexNAcases probably have a +1 subsite,in addition to �1 subsite, to stabilize the +1 sugar of the substrate,whereas lysosomal HexNAcases such as the human enzymes haveonly the �1 subsite. From their results, they concluded that themechanism of selective inhibition of 1 was related to the existenceof the +1 subsite. Furthermore, Yu and co-workers synthesized aseries of analogs of 1 and explained the selective inhibition mech-anism of 1 based on inhibitory kinetics and molecular dockingusing these analogs and several HexNAcases.8 They presumed thatthe GlcNAc component next to TMG binds at the +1 subsite andstabilizes the binding conformation of TMG bound to �1 subsitein TMG-chitotrimycin-sensitive enzymes. These results confirmedthe correlation between the inhibitory specificity of 1 and the sub-strate specificity of HexNAcases. To our knowledge, however, thereis no previous study demonstrating the practical use of 1 as a probefor their substrate specificity and physiological functions of Hex-NAcases. The present work was conducted to examine the feasibil-ity of using TMG-chitotriomycin (1) as a probe for the prediction ofsubstrate specificity, especially focusing on the distinction be-tween enzymes involved in chitin degradation and those involvedin glycoconjugate processing.

Fifteen HexNAcases from various organisms were tested for theirsensitivity toward 1 and the well-studied inhibitors, PUGNAc (2)and NAG-thiazoline (3). The result is presented in Table 1. PUGNAc

Figure 1. Chemical structures of TMG-chitotriomycin (1), PUGNAc (2), and NAG-thiazoline (3).

Table 1Inhibitory activity of TMG-chitotriomycin (1), PUGNAc (2), and NAG-thiazoline (3).

Origin of enzymes (enzyme name) Family PUGNAc NAG-thiazoline TMG-chitotrio-mycinIC50 (lM) IC50 (lM) IC50 (lM)

Streptomyces coelicolor (SCO2758) Bacterium 3 1.80 >1000 >70.0Spodoptera litura (crude enzyme) Insect NA 0.762 28.4 0.526Aspergillus niger Fungus NA 1.24 34.0 3.66Aspergillus oryzae Fungus NA 0.492 113 1.46Penicillium oxalicum Fungus NA 0.970 17.3 0.439Streptomyces plicatus (SpHex) Bacterium 20 0.0148 44.1 14.7Streptomyces griseus (SGR4591) Bacterium 20 0.541 27.6 >70.0Streptococcus pneumoniae Bacterium NA 0.0034 1.65 >70.0Jack bean Plant NA 0.679 3.55 >70.1Bovine kidney Mammal NA 0.119 1.37 >70.1Human placenta Mammal NA 0.0453 0.545 >70.1Porcine placenta (GlcNAcase A) Mammal NA 0.0753 1.47 >70.0Turbo cornutus Mollusk NA 0.0739 0.212 >70.0Bombyx mori (BmGlcNAcase2) Insect 20 1.69 0.909 >70.0Bombyx mori (BmFDL) Insect 20 2.01 527 >70.0

NA: not assigned (GH family assignment has not been reported).

30 H. Shiota et al. / Carbohydrate Research 375 (2013) 29–34

(2) inhibited all tested HexNAcases (IC50 = 0.0034–2.01 lM) andNAG-thiazoline (3) inhibited HexNAcases (IC50 = 0.212–527 lM)other than that from Streptomyces coelicolor (SCO2758) belongingto GH3 family. In contrast, TMG-chitotriomycin (1) specificallyinhibited five HexNAcases from Spodoptera litura, Aspergillus niger,Aspergillus oryzae, Penicillium oxalicum, and Streptomyces plicatus(IC50 = 0.439–14.7 lM). Inhibitor 3 is known as a specific inhibitoragainst GH20 and GH84 HexNAcases. Considering the limited distri-bution of GH84 HexNAcases, all enzymes inhibited by 3 in this workwere assumed to belong to GH20. Therefore, it was indicated thatGH20 HexNAcases were subdivided into 1-sensitive and 1-insensi-tive enzymes. HexNAcases from Aspergillus oryzae, Penicilliumoxalicum, and Streptomyces plicatus were selected as the representa-tives of the 1-sensitive enzymes, and HexNAcases from Streptomycesgriseus, Jack bean, and Human placenta were selected as the repre-sentatives of the 1-insensitive HexNAcases. Their substrate specific-ity was examined with attention focusing on the distinctionbetween enzymes involved in chitin degradation and those involvedin glycoconjugate processing.

First, the 1-sensitive and 1-insensitive HexNAcases were exam-ined for hydrolytic activity toward p-nitrophenyl-derivatized N-acetylchitooligosaccharides and underivatized N-acetylchitooligo-saccharides. The 1-sensitive HexNAcases (enzymes from A. oryzae,P. oxalicum, and S. plicatus) liberated p-nitrophenol from all p-

nitrophenyl-derivatized N-acetylchitooligosaccharides tested (Glc-NAc-pNP, GlcNAc2-pNP, and GlcNAc3-pNP), while the 1-insensitiveHexNAcases (enzymes from jack bean, human placenta, and S. gris-eus) scarcely hydrolyzed GlcNAc2-pNP and GlcNAc3-pNP (Fig. 2).Moreover, the 1-sensitive HexNAcases showed hydrolytic activitytoward underivatized N-acetylchitooligosaccharide (GlcNAc2-4)(Fig. 3) but the 1-insensitive HexNAcases showed no activity to-ward these underivatized N-acetylchitooligosaccharides includingthe disaccharide GlcNAc2 (data not shown). These results indicatethat the 1-sensitive HexNAcases prefer underivatized N-acetylchi-tooligosaccharides and p-nitrophenyl-derivatized N-acetylchitooli-gosaccharides as substrates while the 1-insensitive HexNAcases donot. This view is supported by the results of Iwamoto et al. who re-ported that HexNAcase from A. niger (the 1-sensitive HexNAcase inthis work) hydrolyzed underivatized N-acetylchitooligosaccharides(GlcNAc2, GlcNAc3) and p-nitrophenyl-derivatized N-acetylchitool-igosaccharides (GlcNAc-pNP, GlcNAc2-pNP), while jack bean Hex-NAcase (the 1-insensitive HexNAcase in this work) did nothydrolyze these substrates except for GlcNAc-pNP.9

The 1-sensitive and 1-insensitive HexNAcases were investi-gated further for substrate specificity. HexNAcases from jack bean,Streptococcus pneumoniae and bovine kidney are reported to liber-ate non-reducing-end GlcNAc residues from N-glycans, and humanplacenta HexNAcases are reported to recognize GM2 gangliosides

0 10 20 30 40Relative hydrolysis activity (%)

(the percentage of pNP-releasing activity relative to that toward GlcNAc-pNP)

Aspergillusoryzae

Penicilliumoxalicum

Streptomycesplicatus

Streptomycesgriseus

Jack bean

Human placenta

GlcNAc2-pNPGlcNAc3-pNP

1-Se

nsiti

veH

exN

Aca

ses

1-In

sens

itive

H

exN

Aca

ses

Figure 2. Relative hydrolysis activity of TMG-chitotriomycin-sensitive and TMG-chitotriomycin-insensitive HexNAcases toward p-nitrophenyl N-acetylchitooligosaccha-rides. TMG-chitotriomycin-sensitive and TMG-chitotriomycin-insensitive HexNAcases were incubated for 1 h with 0.5 mM GlcNAcn-pNP at 37 �C. The hydrolytic activity ofeach enzyme was determined by measuring the amount of p-nitrophenol released at 415 nm after the addition of 1.3 M NaOH. The relative hydrolysis activity towardGlcNAc2-pNP and GlcNAc3-pNP for each enzyme is expressed as the percentage of activity relative to that toward GlcNAc-pNP (set to 100%).

Figure 3. Time-course of hydrolysis of GlcNAc2 (A), GlcNAc3 (B), and GlcNAc4 (C) catalyzed by HexNAcase from Aspergillus oryzae (TMG-chitotriomycin-sensitive HexNAcase).HexNAcase from Aspergillus oryzae was incubated with each substrate (2.5 mM) at 37 �C. Samples were then analyzed by HPLC with UV detection at 210 nm.

H. Shiota et al. / Carbohydrate Research 375 (2013) 29–34 31

as substrates.10–12 Therefore, the 1-insensitive HexNAcases werethought to recognize the glycan moiety of glycoconjugates as sub-strates. To prove this, the 1-sensitive and 1-insensitive HexNAcaseswere examined for hydrolytic activity toward 2-aminopyridine(PA)-derivatized GnGn, a biantennary core structure of the com-plex type N-glycan chains. HPLC analysis of reaction mixturesshowed that the 1-insensitive HexNAcases hydrolyzed GnGn-PAto MM-PA through GnM-PA and/or MGn-PA by cleaving off the ter-minal b1,2)-linked GlcNAc residues, while the 1-sensitive HexNA-cases did not hydrolyze it (Fig. 4). These results suggest that the1-insensitive HexNAcases accept the branched sugar chain sub-strates such as N-glycans to participate in glycoconjugate process-ing, but such is not the case with the 1-sensitive HexNAcases. Theactive pockets of the 1-insensitive HexNAcases are regarded as suf-ficiently large to accommodate sterically hindered branching sub-strates, whereas those of the 1 sensitive HexNAcases are not.

Our results demonstrate that 1 would be used as a probe for theprediction of substrate specificity of HexNAcases. Substrate speci-ficity of HexNAcases is closely related to their physiological func-tions, mainly chitin degradation and glycoconjugate processing,indicating that 1 would be used also as a probe for the predictionof physiological functions of uncharacterized HexNAcases. Forexample, both SpHex and SGR4591 are HexNAcases from the acti-nomycetes belonging to the genus Streptomyces, but the formerwas 1-sensitive and the latter was 1-insensitive, indicating thatSpHex and SGR4591 are involved respectively in chitin degrada-tion and glycoconjugate processing. Similarly, the crude HexNA-case from Spodoptera litura and the HexNAcases from Bombyx

mori (BmGlcNAcase2 and BmFDL) are the enzymes of lepidopteraninsects, but the former was 1-sensitive and the latter was 1-insen-sitive, indicating that HexNAcase (s) in the crude enzyme from S.litura is/are involved in chitin degradation and that BmGlcNAcase2and BmFDL are involved in glycoconjugate processing. Indeed,BmFDL is suggested to be an N-glycan processing HexNAcase inB. mori.13 It is particularly interesting that BmGlcNAcase2 canhydrolyze N-acetylchitotriose (GlcNAc3) to N-acetylchitobiose(GlcNAc2) in addition to N-glycan trimming ability14 although itis 1-insensitive. Recently, Kokuho et al. reported that BmGlcNA-case2 (designated as ‘BmGlcNAcase isoform A’ in their paper)was found to share genetic, and possibly, functional propertieswith the novel HexNAcase they found (designated as ‘BmGlcNA-case 2 isoform A’ in their paper), which is suggested to play uniquefunctions such as decomposition (or detoxification) of feed ingredi-ents and enhancement of the assembly of intrinsic, possibly, highmannose-type carbohydrates.15 This unique substrate specificitymight result from ‘functional shifts’ in evolution as well as the in-sect HexNAcases encoded by the fused lobes (fdl) gene, which ap-pear to be derived from exochitinase-like ancestors but which nowplay a role in the metabolism of paucimannosidic N-glycans.1

Due to lack of availability, only one HexNAcase (enzyme fromStreptomyces coelicolor (SCO2758)) belonging to GH3 HexNAcasewas tested for the sensitivity toward 1 in the present work. Thisenzyme was found to be 1-insensitive, but the physiological roleof this enzyme is not known. Therefore, it remains to be clarifiedwhether other GH3 HexNAcases are 1-insensitive or not. The sen-sitivity of NAG A from Streptomyces thermoviolaceus, which is

Figure 4. Assay for the hydrolysis of GnGn-PA by TMG-chitotriomycin-sensitive and TMG-chitotriomycin-insensitive HexNAcases. TMG-chitotriomycin-sensitive (A) and -insensitive (B) HexNAcases were incubated for 16 h with 250 nM GnGn-PA at 37 �C. Samples were then analyzed by HPLC with fluorescence detection. (C) The structure ofGnGn-PA.

32 H. Shiota et al. / Carbohydrate Research 375 (2013) 29–34

assumed to be involved in chitin degradation,16 is of particularinterest.

In conclusion, results show that that TMG-chitotriomycin (1)can be used not only to predict the substrate specificity of HexNA-cases but also to predict their functions. In addition, very recentlyHalila et al. have developed a straightforward chemo-biotechno-logical approach for the rapid production of TMG-chitotriomycin,17

indicating that 1 may become an easily accessible probe.

1. Experimental

1.1. General methods

b-N-Acetylhexosaminidase from Spodoptera litura was preparedas described in our previous papers.18,19 b-N-Acetylhexosaminidas-es from Aspergillus oryzae, Aspergillus niger, jack bean (Canavaliaensiformis), bovine kidney, human placenta, and porcine placentaand PUGNAc were purchased from Sigma–Aldrich. b-N-Acetylh-exosaminidases from Penicillium oxalicum, Streptococcus pneumoniaand Turbo cornatus, underivatized N-acetylchitooligosaccharides(GlcNAc1–6) and p-nitrophenyl-derivatized N-acetylchitooligosac-charides (GlcNAc1–3-pNP) were purchased from Seikagaku Kogyo(Tokyo, Japan). b-N-Acetylhexosaminidase from Streptomyces plica-tus (SpHex) was purchased from New England Biolabs (Ipswich,MA, USA). b-N-Acetylhexosaminidases from Bombyx mori(BmGlcNAcase2 and BmFDL) were kindly provided by Sysmex Cor-poration (Kobe, Japan). TMG-chitotriomycin was prepared as de-scribed previously.5 NAG-thiazoline was synthesized fromperacetylated GlcNAc using Lawesson’s reagent according to theprocedure of Knapp et al.20 and Reid et al.21 withmodifications. GnGn-PA [(GlcNAcb1,2Mana1,6)(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4GlcNAc-PA] and MM-PA [Mana1,6(Mana1,3)Manb1,4GlcNAcb1,4GlcNAc-PA]were purchased from

Takara Bio (Shiga, Japan). GnM-PA [(Glc-NAcb1,2Mana1,6)(Mana1,3)Manb1,4GlcNAcb1,4GlcNAc-PA] andMGn-PA [Mana1,6(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4Glc-NAc-PA] were purchased from the Masuda Chemical Industry(Kagawa, Japan). All other reagents were commercially available.

1.2. Cloning, overexpression, and purification of HexNAcasesfrom Streptomyces coelicolor (SCO2758) and Streptomycescoelicolor (SGR4591)

Cloning, overexpression, and purification of GH3 HexNAcaseSCO2758 and GH20 HexNAcase SGR4591 were carried out as de-scribed previously.22,23 Briefly, genomic DNAs were prepared fromStreptomyces coelicolor A3 (2)24 or Streptomyces griseus IFO1335025

using the method described by Hopwood et al.26 Each gene encod-ing SCO2758 or SGR4591 (http://strepdb.streptomyces.org.uk/)was amplified using PCR with a set of sense primers incorporatingthe NdeI site upstream of a start codon (5-ATA-TATATCATATGCACCACAGCAGCACGGC-3 for SCO2758 and 5-ATA-TATATCATATGACACCCGCGAACTCCGT-3 for SGR4591), the anti-sense primer incorporating the HindIII site downstream of a stopcodon (5-ATATAAGCTTCTACGACCGGTAGGTCAGCC-3 for SCO2758and 5-ATATAAGCTTCACACCACGGGCGGCGCGC-3 for SGR4591),and the genomic DNA. In addition, a set of Prime Star GXL andPrime Star GXL buffers (Takara) was used for the PCR reaction.The PCR products were digested using NdeI and HindIII to obtainthe gene fragments encoding SCO2758 or SGR4591.

The fragments encoding SCO2758 were used for the construc-tion of the final expression plasmid as follows. The Streptomyceshyperexpression system27 was adopted for the overexpression ofthe enzyme. In brief, the above fragment was introduced into theNdeI/HindIII gap of pTONA5a to generate the expression plasmid,after which it was transferred from Escherichia coli to

H. Shiota et al. / Carbohydrate Research 375 (2013) 29–34 33

S. lividans1326. The fragments encoding SGR4591 were used forthe construction of the final expression plasmid (pET28-His6-SGR4591) as described previously using expression vectorsdesigned for the N-terminal His6-tag.28–30 An overnight expresssystem 1 (Novagen Inc., Germany) and Rosetta 2 (DE3) were usedfor the overexpression of the enzyme as described previously.28–30

HexNAcase SCO2758 was secreted at high levels in the culturebroth. The culture filtrate was dialyzed followed by analysis usingsodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under a reducing condition with Coomassie Blue staining.HexNAcase SCO2758 was obtained as almost pure, and therefore,the dialyzed fraction was used as the purified enzyme. HexNAcaseSGR4591 was purified from the sonicated cell lysis using metalaffinity resin (Talon; Clontech, Japan) according to the manufac-turer’s instructions. The recombinant proteins were washed with5 mM imidazole, and then eluted with 100 mM imidazole. The elu-ate was concentrated and dialyzed, and then used as the purifiedenzyme.

1.3. HexNAcase inhibition assays

Inhibitory activity (IC50 value) was determined by a colorimet-ric method of our previous work5,19 using 0.5 mM p-nitrophenyl b-N-acetylglucosaminide (GlcNAc-pNP) as a substrate and 1.13 units/lL of each HexNAcase. The release of p-nitrophenol was measuredat 415 nm after the addition of 1.3 M NaOH. IC50 values were cal-culated by plotting the inhibitor concentration versus the rate ofinhibition. One unit of enzyme was defined as the amount of en-zyme that liberates 1 lmol of p-nitrophenol per min at 37 �C undereach assay condition in which H2O was used instead of the inhib-itor solution. The following buffers were used according to theinstructions or references: (1) Spodoptera litura19 and BmFDL (Bom-byx mori)13: 100 mM citrate–phosphate–borate buffer (pH 6.0), (2)Aspergillus oryzae, jack bean, and bovine kidney: 100 mM citratebuffer (pH 5.0) containing 100 mM NaCl and 0.01% (w/v) bovineserum albumin, (3) human placenta: 100 mM citrate buffer (pH4.5) containing 100 mM NaCl and 0.01% (w/v) bovine serum albu-min, (4) Aspergillus niger and Streptococcus pneumonia: 100 mM cit-rate buffer (pH 5.0), (5) Penicillium oxalicum, Streptomyces plicatus,and porcine placenta: 100 mM citrate buffer (pH 4.5), (6) Turbocornatus and Bombyx mori (BmGlcNAcase2):16 100 mM citrate–phosphate–borate buffer (pH 4.5), (7) Streptomyces coelicolor(SCO2758): 100 mM citrate–phosphate–borate buffer (pH 6.3)and (8) Streptomyces griseus (SGR4591): 100 mM citrate–phos-phate–borate buffer (pH 6.7).

1.4. Assay for hydrolysis of various substrates by 1-sensitive and1-insensitive HexNAcases

1-Sensitive HexNAcases (enzymes from A. oryzae, P. oxalicum,and S. plicatus) and 1-insensitive HexNAcases (enzymes from jackbean, human placenta, and S. griseus) were examined for hydrolyticactivity toward various substrates. In each HexNAcase assay fol-lowing buffers were used: (1) A. oryzae and Jack bean: 100 mM cit-rate buffer (pH 5.0), (2) P. oxalicum, S. plicatus and human placenta:100 mM citrate buffer (pH 4.5), (3) S. griseus (SGR4591): 100 mMcitrate–phosphate–borate buffer (pH 6.7).

1.4.1. Assay for hydrolysis of p-nitrophenyl N-acetylchitooligosaccharides

Hydrolytic activity toward p-nitrophenyl N-acetylchitooligosac-charides (GlcNAc1–3-pNP) was determined by the method de-scribed in Section 3 except for the addition of H2O instead ofinhibitor solution to reaction mixture. In this assay, 11.3 units/lL

of each HexNAcase was used. If the absorbance (A415) exceeded1.0, the reaction mixture was diluted with H2O until the absor-bance is less than 1.0 in order to ensure linearity.

1.4.2. Assay for hydrolysis of N-acetylchitooligosaccharidesHydrolytic activity toward unmodified N-acetylchitooligosac-

charides (GlcNAc1–4) was measured by the HPLC-UV method. Thereaction mixture contained enzyme solution, 100 mM assay bufferand 2.5 mM substrate in a total volume of 200 lL. After incubatingthe reaction mixture at 37 �C for an appropriate period, a portion ofthe reaction mixture was withdrawn, mixed with twice the volumeof acetonitrile to quench the reaction, and adjusted the concentra-tion of acetonitrile to 67%, which is close to that of acetonitrile(70%) in HPLC mobile phase below. The solution was then dilutedfourfold with 67% acetonitrile to adjust the concentration of N-acetylchitooligosaccharides. This solution was centrifuged andthe supernatant was analyzed by the normal phase HPLC using aAsahipak NH2P50-4E column (u 4.6 � 250 mm, Showa Denko, To-kyo). The elution was conducted using 70% acetonitrile at a flowrate of 1.0 mL/min with UV detection at 210 nm. The column wasmaintained in a column oven at 40 �C. N-Acetylchitooligosaccha-ride concentration at each reaction time was determined by thestandard curb obtained with an authentic sample of N-acetylchi-tooligosaccharide mixture (Seikagaku Kogyo, Tokyo). Enzyme solu-tion of 56.5 units/lL was used for HexNAcases from A. oryzae, P.oxalicum, and S. plicatus. Enzyme solution of 565 units/lL was usedfor HexNAcases from Jack bean, human placenta, and S. griseus.

1.4.3. Assay for hydrolysis of GnGn-PAHydrolytic activity toward an N-glycan core structure was

determined using GnGn-PA as a substrate. The reaction mixturecontaining enzyme solution, 100 mM assay buffer, and 250 nMGnGn-PA in a total volume of 100 lL was used. After incubatingat 37 �C for 16 h, the reaction mixture was boiled at 95 �C for5 min and centrifuged. The supernatant was analyzed by reversedphase HPLC with fluorescence detection (Ex = 310 nm,Em = 380 nm). HPLC analysis was carried out according to themethod of Okada et al.14 with the following modifications. The col-umn was an Inertsil ODS-3 column (u 4.6 � 250 mm, GL Science)and the flow rate was 0.7 mL/min. Peaks corresponding to thehydrolytic products were identified by comparison of retentiontimes with authentic samples of MM-PA, GnM-PA, and MGn-PA.Enzyme solution of 56.5 units/lL was used for HexNAcase fromjack bean. Enzyme solution of 565 units/lL was used for HexNA-cases from human placenta, S. griseus, and P. oxalicum. Enzymesolution of 5650 units/lL was used for HexNAcases from A. oryzae,and S. plicatus.

Acknowledgements

The authors would like to thank Sysmex Corporation (Kobe, Ja-pan) for generous gifts of BmGlcNAcase and BmFDL. The authorswould like to thank Dr. Kazuhito Fujiyama (Osaka University) forhelpful advice. This work was supported by MEXT/JSPS KAKENHI‘Grant-in-Aid for Scientific Research (C), 21580128’ to T.N.

References

1. Intra, J.; Perotti, M. E.; Pavesi, G.; Horner, D. S. BMC Evol. Biol. 2008, 8, 214.2. Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat,

B. Nucleic Acids Res. 2009, 37, D233.3. Slamova, K.; Bojarova, P.; Petraskova, L.; Kren, V. Biotechnol. Adv. 2010, 28, 682.4. Scaffidi, A.; Stubbs, K. A.; Dennis, R. J.; Taylor, E. J.; Davies, G. J.; Vocadlo, D. J.;

Stick, R. V. Org. Biomol. Chem. 2007, 5, 3013.5. Usuki, H.; Nitoda, T.; Ichikawa, M.; Yamaji, N.; Iwashita, T.; Komura, H.;

Kanzaki, H. J. Am. Chem. Soc. 2008, 130, 4146.6. Yang, Y.; Li, Y.; Yu, B. J. Am. Chem. Soc. 2009, 131, 12076.

34 H. Shiota et al. / Carbohydrate Research 375 (2013) 29–34

7. Liu, T.; Zhang, H.; Liu, F.; Wu, Q.; Shen, X.; Yang, Q. J. Biol. Chem. 2011, 286,4049.

8. Yang, Y.; Liu, T.; Yang, Y.; Wu, Q.; Yang, Q.; Yu, B. ChemBioChem 2011, 3, 457.9. Iwamoto, T.; Sonobe, A.; Somsit, A.; Nagasaki, S. Biosci. Biotechnol. Biochem.

1993, 57, 841.10. Gutternigg, M.; Ahrer, K.; Grabher-Meier, H.; Bürgmayr, S.; Staudacher, E. Eur. J.

Biochem. 2004, 271, 1348.11. Gutternigg, M.; Kretschmer-Lubich, D.; Paschinger, K.; Rendic, D.; Hader, J.;

Geier, P.; Ranftl, R.; Jantsch, V.; Lochnit, G.; Wilson, I. B. H. J. Biol. Chem. 2007,282, 27825.

12. Akeboshi, H.; Chiba, Y.; Kasahara, Y.; Takashiba, M.; Takaoka, Y.; Ohsawa, M.;Tajima, Y.; Kawashima, I.; Tsuji, D.; Itoh, K.; Sakuraba, H.; Jigami, Y. Appl.Environ. Microbiol. 2007, 73, 4805.

13. Nomura, T.; Ikeda, M.; Ishiyama, S.; Mita, K.; Tamura, T.; Okada, T.; Fujiyama,K.; Usami, A. J. Biosci. Bioeng. 2010, 110, 386.

14. Okada, T.; Ishiyama, S.; Sezutsu, H.; Usami, A.; Tamura, T.; Mita, K.; Fujiyama,K.; Seki, T. Biosci. Biotechnol. Biochem. 2007, 71, 1626.

15. Kokuho, T.; Yasukochi, Y.; Watanabe, S.; Inumaru, S. Genes Cells 2010, 15, 525.16. Tsujibo, H.; Hatano, N.; Mikami, T.; Hirasawa, A.; Miyamoto, K.; Inamori, Y.

Appl. Environ. Microbiol. 1998, 64, 2920.17. Halila, S.; Samain, E.; Vorgias, C. E.; Armand, S. Carbohydr. Res. 2013, 368, 52.18. Nitoda, T.; Usuki, H.; Kanzaki, H. Z. Naturforsch. 2003, 58c, 891.19. Usuki, H.; Nitoda, T.; Okuda, T.; Kanzaki, H. J. Pestic. Sci. 2006, 31, 41.20. Knapp, S.; Vocadlo, D.; Gao, Z.; Kirk, B.; Lou, J.; Withers, S. G. J. Am. Chem. Soc.

1996, 118, 6804.21. Reid, C. W.; Blackburn, N. T.; Clarke, A. J. FEMS Microbiol. Lett. 2004, 234, 343.

22. Usuki, H.; Yamamoto, Y.; Kumagai, Y.; Nitoda, T.; Kanzaki, H.; Hatanaka, T. Org.Biomol. Chem. 2011, 9, 2943.

23. Usuki, H.; Yamamoto, Y.; Arima, J.; Iwabuchi, M.; Miyoshi, S.; Nitoda, T.;Hatanaka, T. Org. Biomol. Chem. 2011, 9, 2327.

24. Bentley, S. D.; Chater, K. F.; Cerdeno-Tarraga, A. M.; Challis, G. L.; Thomson, N.R.; James, K. D.; Harris, D. E.; Quail, M. A.; Kieser, H.; Harper, D.; Bateman, A.;Brown, S.; Chandra, G.; Chen, C. W.; Collins, M.; Cronin, A.; Fraser, A.; Goble, A.;Hidalgo, J.; Hornsby, T.; Howarth, S.; Huang, C. H.; Kieser, T.; Larke, L.; Murphy,L.; Oliver, K.; O’Neil, S.; Rabbinowitsch, E.; Rajandream, M. A.; Rutherford, K.;Rutter, S.; Seeger, K.; Saunders, D.; Sharp, S.; Squares, R.; Squares, S.; Taylor, K.;Warren, T.; Wietzorrek, A.; Woodward, J.; Barrell, B. G.; Parkhill, J.; Hopwood,D. A. Nature 2002, 417, 141.

25. Ohnishi, Y.; Ishikawa, J.; Hara, H.; Suzuki, H.; Ikenoya, M.; Ikeda, H.; Yamashita,A.; Hattori, M.; Horinouchi, S. J. Bacteriol. 2008, 190, 4050.

26. Hopwood, D. A.; Bibb, M. J.; Chater, K. F.; Kieser, T.; Bruton, C. J.; Kieser, H. M.;Lydiate, D. J.; Smith, C. P.; Ward, J. M.; Schrempf, H. A Laboratory Manual; TheJohn Ines Foundation: Norwich, UK, 1985.

27. Hatanaka, T.; Onaka, H.; Arima, J.; Uraji, M.; Uesugi, Y.; Usuki, H.; Nishimoto, Y.;Iwabuchi, M. Protein Expr. Purif. 2008, 62, 244.

28. Usuki, H.; Uesugi, Y.; Arima, J.; Yamamoto, Y.; Iwabuchi, M.; Hatanaka, T. Chem.Commun. 2010, 580.

29. Usuki, H.; Uesugi, Y.; Iwabuchi, M.; Hatanaka, T. Biochim. Biophys. Acta, ProteinsProteomics 2009, 1794, 468.

30. Usuki, H.; Uesugi, Y.; Iwabuchi, M.; Hatanaka, T. Biochim. Biophys. Acta, ProteinsProteomics 2009, 1794, 1673–1683.