Improved fluorescent labeling of chitin oligomers: Chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions

Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing. AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH 8.

jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / c a r b p o l

conditions

Satoshi Wakitaa,1, Masahiro Kimuraa,1, Naoki Katoa, Akinori Kashimuraa,

Shunsuke Kobayashia, Naoto Kanayamaa, Misa Ohnoa, Shotaro Hondaa,

Masayoshi Sakaguchia, Yasusato Sugaharaa, Peter O Bauerb,c, Fumitaka Oyamaa,∗

a Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan

b Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA

c Bioinova Ltd., Prague 142 20, Czechia

a r t i c l e i n f o

Article history:

Received 14 June 2016

Received in revised form 3 January 2017

Accepted 29 January 2017

Available online 31 January 2017

Keywords:

Acidic mammalian chitinase

Chitin

Chitin degradation products

Chitin oligomers

Fluorophore

Pre-acidification method

a b s t r a c t Acidicmammalianchitinase(AMCase)hasbeenimplicatedinvariouspathophysiologicalconditions includingasthma,allergicinflammationandfoodprocessing.AMCaseismostactiveatpH2.0,andits activitygraduallydecreasestouptopH8.HereweanalyzedchitindegradationbyAMCaseinweak acidictoneutralconditionsbyfluorophore-assistedcarbohydrateelectrophoresisestablishedoriginally foroligosaccharidesanalysis.Wefoundthatspecificfragmentswithslower-than-expectedmobilityas definedbychitinoligosaccharidemarkersweregeneratedatpH5.0∼8.0asby-productsofthereaction

Weestablishedanimprovedmethodforchitinoligosaccharidessuppressingthissidereactionby pre-acidificationofthefluorophore-labelingreactionmixture.Ourimprovedmethodspecificallydetects chitinoligosaccharidesandwarrantsquantificationofupto50nmolofthematerial.Usingthisstrategy,

wefoundthatAMCaseproduceddimerofN-acetyl-d-glucosamine(GlcNAc)atstrongacidictoneutral condition.Moreover,wefoundthatAMCasegenerates(GlcNAc)2aswellas(GlcNAc)3underphysiological conditions

©2017TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1 Introduction

Chitinisa ␤-1,4-linked polymer,insolublein mostsolvents,

composedprimarilyofN-acetyl-d-glucosamine(GlcNAc)residues

Itis amajorcomponentoftheexoskeletonsof crustaceansand

insects,themicrofilarialsheathsofparasiticnematodesandfungal

cellwalls(Khoushab&Yamabhai,2010;Koch,Stougaard,&Spaink,

2015).Thus,chitinisthesecondmostabundantpolysaccharidein

nature

Chitinasesareglycosidasesthatbreakdownglycosidicbonds

in chitin They are important enzymes responsible for chitin

metabolisminawiderangeoforganisms,includingbacteria,fungi,

nematodesandarthropods(Bueter,Specht,&Levitz,2013;Hamid

etal.,2013;Khoushab&Yamabhai,2010;Kochetal.,2015;Lee

etal.,2011).Althoughmammalsdonotproducechitin,miceand

∗ Corresponding author.

E-mail address: f-oyama@cc.kogakuin.ac.jp (F Oyama).

1 These authors contributed equally to this article.

humansexpresstwoactivechitinases,chitotriosidase(Chit1)and acidicmammalianchitinase(AMCase)(Bussink,vanEijk,Renkema, Aerts,&Boot,2006;Leeetal.,2011).Chit1wasthefirstmammalian chitinasetobepurifiedanditsgenewascloned(Boot,Renkema, Strijland,vanZonneveld,&Aerts,1995;Renkema,Boot,Muijsers, Donker-Koopman,&Aerts,1995).AMCasewasthesecond mam-malianchitinasediscoveredandwasnamedforitsacidicisoelectric point(Bootetal.,2001)

AMCasehasattractedconsiderableattentionduetoitsincreased expression under certain pathological conditions related to immuneresponse,forexampleinaninducedasthmamousemodel andantigen-inducedmousemodelsofallergiclunginflammation (Reeseetal.,2007;Zhuetal.,2004).Somepolymorphismsand hap-lotypesintheAMCasegeneareassociatedwithbronchialasthma

inhumans(Bierbaumetal.,2005;Okawaetal.,2016;Seiboldetal.,

2009)andinhibitionofitsactivityhasbeensuggestedasa thera-peuticstrategyagainstasthma(Sutherlandetal.,2011;Yangetal.,

2009).Furthermore,AMCasehasbeenshowntobeinvolvedineye (Bucolo,Musumeci,Maltese,Drago,&Musumeci,2008; Bucolo,

http://dx.doi.org/10.1016/j.carbpol.2017.01.095

0144-8617/© 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.

stomachdiseases(Cozzarinietal.,2009;Nookaewetal.,2013)

We havereported that AMCasemRNAis synthesizedin the

mousestomachatexceptionallyhighlevels.Theselevelsare

com-parabletopepsinogen,theprecursorofthemajordigestiveenzyme

in gastric fluid, pepsin, suggesting a digestive role of AMCase

(Ohnoetal.,2013; Ohno,Tsuda,Sakaguchi,Sugahara,&Oyama,

2012).Moreover,werecentlyshowedthatAMCaseisa

proteases-resistantglycosidaseinmousedigestivesystem,furthersupporting

thehypothesisofAMCasefunctioningasadigestiveenzyme(Ohno

etal.,2016).Wehavealsoshownthatbesidestomach,AMCase

mRNAishighlyexpressedinsubmaxillaryglandandlung(Ohno

etal., 2012).In addition,recombinant AMCaseand its catalytic

domainhad thehighestactivityataroundpH2.0,whenit

pro-ducesprimarily(GlcNAc)2,andloweractivitiesatmoreneutralpH

(pH3.0∼7.0)(Bootetal.,2001;Kashimuraetal.,2015;Kashimura

etal.,2013).TheAMCaseactivityundersomatictissueconditions

atpH5∼8remainstobeelucidated

Chitin and chitosan oligosaccharides

(N-acetyl-chitooligosaccharides) prepared either chemically or

enzymatically, have been shown to have anti-cancer and

anti-inflammatory properties (Azuma, Osaki, Minami, & Okamoto,

2015;Masudaetal.,2014)andhavevariousbiologicalactivities

in mammalian cells (Aam, Heggset, Norberg, Sorlie, Varum, &

Eijsink, 2010; Khoushab & Yamabhai, 2010) We hypothesized

that upregulated AMCaseunder certain pathological conditions

cangeneratespecificdegradationproductsassociatedwiththose

pathologies

HereweanalyzedthechitinaseactivityofAMCaseby

incubat-ingthe enzyme withchitin substratesat pH2.0∼8.0 followed

by fluorophore-assisted carbohydrate electrophoresis (FACE), a

methodbasedonlabelingthereducingendsofoligosaccharides

withafluorophore(Jackson,1990).FACEisverysensitive(pmol

amounts)ascomparedtohigh-performanceliquid

chromatogra-phy(HPLC)andnuclearmagneticresonance(NMR)spectrometry,

andisoftenusedfordetectionofverylowoligosaccharide

quanti-ties(Bootetal.,2001;Jackson,1990)

Duringourresearch,wefoundapH-dependentgenerationof

anunexpectedby-productwithaslowermobilitythan(GlcNAc)2,

themainfragmentresultingfromchitinsubstratesdigestionby

AMCase.Thisby-productwasobservedatpH5.0∼8.0.Tooptimize

thedigestionreaction,weestablishedanimprovedmethodfora

specificdetectionofchitinoligosaccharides.Usingthisprocedure,

wefoundthatAMCasegenerates(GlcNAc)2atbroadpHrangeof

2.0∼8.0

2 Materials and methods

2.1 RecombinantAMCaseexpressedinEscherichiacoliand

enzymaticactivityassays

We expressed and purified Protein A-AMCase-V5-His from

the periplasmic fraction of the E coli as described previously

(Kashimura et al., 2015; Kashimura et al., 2013).The

protein-containingfractionsweredesaltedusing PDMidiTrapG-25 (GE

Healthcare, Milwaukee, WI, USA) equilibrated with TS buffer

[20mMTris-HCl(pH7.6),150mMNaClandproteininhibitor

(Com-pleteMini;RocheDiagnostics,Basel,Switzerland)]

Chitinolyticactivitywasdetermined using a synthetic

chro-mogenicsubstrate, 4-nitrophenyl N,N-diacetyl-␤-d-chitobioside

(Sigma-Aldrich, St Louis, MO, USA) as described previously

(Kashimuraetal.,2015;Kashimuraetal.,2013).AMCaseunit

defi-nitionwasalsoreportedpreviously(Kashimuraetal.,2013)

2.2 Degradationofcolloidalchitinand(GlcNAc)6byE

coli-expressedmouseAMCase Colloidalchitinwaspreparedfromshrimpshellchitin (Sigma-Aldrich),asdescribedpreviously(Kashimuraetal.,2013),andused

asasubstratetodeterminethechitinaseactivityofAMCase.All enzymaticreactionsusingcolloidalchitin(atafinalconcentration

of 1mg/mL) or N-acetyl-chitohexaose (GlcNAc)6(0.2␮mol/mL) (SeikagakuCorporation,Tokyo,Japan)werecarriedoutina vol-umeof50␮Lcontaining0.8mUor0.1mUE.coli-expressedmouse AMCaseinMcIlvaine’sbuffer(mixtureof0.1Mcitricacidand0.2M

Na2HPO4;pH2.0topH8.0).Thereactionmixtureswereincubated for1hat37◦C

2.3 FluorophorelabelingbythemethodofJackson Generatedchitinfragmentsorchitinmono-andoligomersof (GlcNAc)1 ∼6 (SeikagakuCorporation)asmolecularweight

mark-erswerelabeledcovalentlyattheirreducingendgroupswiththe fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS, Invitrogen, Carlsbad, CA, USA), and separated by 40% poly-acrylamidegel electrophoresis (PAGE), as described by Jackson (Jackson, 1990) Briefly, the enzymatic reaction samples were lyophilized and 5␮Lof 0.2M ANTS in acetic acid/water (3:17, v/v) and 5␮Lof 1.0MNaCNBH3 indimethyl sulfoxide (DMSO) were added The mixture wasincubated at 37◦C for 16h The reaction was neutralized by 10␮L of 1M NaOH, followed by additionof10␮LLaemmlisamplebuffer(Laemmli,1970) with-outSDS,2-mercaptoethanolandbromophenolblue.Thesamples wereseparated byPAGE and quantifiedusing theLuminescent ImageAnalyzer(ImageQuantLAS4000,GEHealthcare),according

tothemanufacturer’sinstructions.Exposureconditionwasfixed

asfollows:exposuretype,precision;sensitivity,highresolution; exposuretime,1s

2.4 Pre-acidificationmethodforlabelingchitinoligomers Enzymaticreactionswerelyophilizedand5␮Lof0.2MANTS

inaceticacid/water(3:17,v/v),5␮Lof1.0MNaCNBH3 inDMSO and5␮Lof17.5Maceticacidwereaddedforreactionacidification followedbyincubationat37◦Cfor16h.Thereactionwas neutral-izedby15␮Lof1MNaOH,followedbyloadingbufferaddition.The sampleswereanalyzedbyPAGEasdescribedabove

2.5 Separationofdegradationproductsfrom(GlcNAc)6by AMCaseusingHPLC

Enzymaticreactionsusing(GlcNAc)6(0.6␮mol/mL)were per-formedinavolumeof300␮Lcontaining4.2mUofE.coli-expressed mouseAMCaseinMcIlvaine’sbuffer(pH2.0orpH7.0),30mM Gly-HCl(pH 2.0)or 30mMTris-HCl(pH7.0).Thereactionmixtures wereincubatedat37◦Cfor1h.GeneratedGlcNAcoligomerswere separatedbygelpermeationchromatography(GPC)essentiallyas describedpreviously(Kazamietal.,2015)

3 Results

3.1 DetectionofpHdependentfluorophore-labeledproductsat

pH5.0∼8.0 Previously,wehaveshownthattheE.coli-producedAMCase hasthe highest chitinolytic activityat around pH 2.0 which is decreasinginless acidicenvironment (pH3.0∼7.0)againstthe syntheticchromogenicsubstrate,4-nitrophenylN,N-diacetyl- ␤-d-chitobioside[4NP-(GlcNAc)2](Kashimuraetal.,2015;Kashimura

etal.,2013).TodeterminewhetherAMCasecangeneratedistinct

withcolloidal chitin or (GlcNAc)6 substratesat pH 2.0∼8.0 in

McIlvaine’sbuffer,followedbyfluorophore-assistedcarbohydrate

electrophoresis(FACE)asoriginallydescribedbyJackson(Jackson,

1990)

AMCasedegradedcolloidalchitinprimarilyto(GlcNAc)2

frag-mentsandtoalesserextentto(GlcNAc)3aswellasstrongdistinct

GlcNAcmonomerunderacidicconditions,whichwereconsistent

withourpreviousobservation(Fig.1A)(Kashimuraetal.,2013)

ThehydrolysiswasevidenttouptopH6.0withdecreasing

activ-ityuponincreasingpH(Fig.1A).Thus,therecombinantAMCase

candegradehighmolecularweightchitinsubstrateatbroadpH

range.However,therewasanatypicalproductgeneratedatpH

5.0∼8.0thatcouldrepresentamobilityshiftofthe(GlcNAc)2

frag-ment(Fig.1Aand B).AMCasealsodegradedalower molecular

weightsubstrate,(GlcNAc)6, andgenerated primarily(GlcNAc)2

andtoalesserextent(GlcNAc)3fragments(Fig.1B).Inaddition,we

detectedtheby-product,whosepresencewasevidentatpHvalues

above3(Fig.1B).TheseresultssuggestapH-dependent

genera-tionofanatypicalby-productfromchitinsubstratesdegradation

byrecombinantAMCaseorfromFACEreaction

3.2 By-productsgenerationinthefluoresceinationreactionand

pre-acidificationprocedurefordetectionoftheGlcNAcoligomers

Next,weexploredwhethertheFACEreactioncouldleadtothe

slowermobilityoftheGlcNAcoligomersintheabsenceofAMCase

at pH2.0 or 7.0 usingMcIlvaine’s buffer Bands withexpected

mobilitywereobtainedwhenincubatedatpH2.0(Fig.2A,leftand

right).Incontrast,incubationatpH7.0resultedintwobandsfrom

eachGlcNAcoligomerusingJacksonmethod(Fig.2A,centerand

right)(Jackson,1990).Slowermigratingby-productsofthe

Glc-NAcoligomersappearedwhenthefluorophorelabelingreaction

wasperformedatpH7.0(Fig.2A).Moreover,thefluoresceination

efficiencywasloweratpH7.0whencomparedtothatatpH2.0

(Fig.2A).Thus,cautionshouldbeexercisedwhenperforming

flu-orescencelabelingoftheGlcNAcoligomersdirectlyobtainedfrom

theenzymaticreactionatpH5.0∼8.0usingMcIlvaine’sbufferby

themethodofJackson(Jackson,1990)

Next, we attempted to optimize the reaction conditions to

suppress the by-product formation at neutral pH When we

acidifiedthereactionwithaceticacidbeforethelabelingwith

fluo-rophore(pre-acidification),weobtainedsinglebandsforalltested

oligomersatbothconditions(Fig.2B).Thus,theby-productswere

formedinapH-dependentmannerduringthefluorescentlabeling

Theseresultsindicatethatourpre-acidificationmethodby

con-centratedaceticacidjustbeforethelabelingreactioneffectively

preventstheformationofunwantedfragments

3.3 Effectofbuffersonthefluorescentlabeling

We next examined the effects of several buffer systems

commonly used in the biochemical evaluation of chitinolytic

activitiesonthefluorescent labelingof (GlcNAc)1∼6 We tested

McIlvaine’s (Fig 3A), phosphate or Tris-HCl (Fig 3B), as well

as 2-(N-morpholino) ethanesulfonic acid (MES),

piperazine-N,N-bis (2-ethanesulfonic acid) (PIPES),

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino)

propanesulfonicacid(MOPS)(Fig.3C)bufferswithfinal

concentra-tionsadjustedto150mMatpH7.0.Ourpre-acidificationmethod

fortheFACEreactionappearedbeneficialirrespectiveoftheused

buffersystems(Fig.3)confirminguniversalenhancementofthe

reactionatpH7.0

3.4 Applicationofthepre-acidificationmethodfor determinationoftheGlcNAcoligomersusingstandardcurve

ToevaluatetheabilityforGlcNAcdimersquantification,we gen-eratedstandardcurvesatpH7.0andfounda linearitybetween (GlcNAc)2andfluorescenceintensitytoupto50nmol(Fig.4).The linearitywaspreservedwiththepre-acidificationstep(Fig.4) ThedetectionsensitivityatpH7.0wasnotinhibitedby increas-ing levelsof aceticacidwhen the (GlcNAc)2 waslabeledusing thepre-acidificationmethod.Thus,ourmethodenablesGlcNAc oligomersquantificationinchitinolyticreactionsperformedatpH 7.0.Toanalyzeasamplethatcontainslessthan1nmolof(GlcNAc)2

per50␮L,thevolumeofthesamplesolutioncouldbemodified from50␮Lto150␮L.Otherwise,additionofconcentratedacetic acidtothesampleorlongerexposureinaluminescentimage ana-lyzerisrecommended

3.5 Re-evaluationofchitinsubstratesdegradationbyAMCase usingpre-acidification

Usingourpre-acidificationmethod,were-evaluatedthe pH-dependentdegradationofchitinsubstratesbyAMCase.Incubation

oftheenzyme(0.8mU)withcolloidalchitinfor1hinMcIlvaine’s bufferatpH2.0∼8.0resultedinproductionofprimarily(GlcNAc)2 witha lowamountof (GlcNAc)3 aswellas strongdistinct Glc-NAcmonomer(Fig.5A).IncubationofAMCasewith(GlcNAc)6for

1hinMcIlvaine’sbufferatpH2.0∼8.0 alsoresultedin produc-tionofprimarily(GlcNAc)2(Fig.5B).ThepH-dependentincrease

of(GlcNAc)3levelswasmorepronouncedascomparedtocolloidal chitin(Fig.5B)

3.6 ChitinolyticpropertiesofAMCaseinweakacidicandneutral conditions

TheresultsdescribedaboveindicatethatAMCasecandegrade colloidalchitinand(GlcNAc)6withrelativelyhighactivityevenin weakacidicandneutralenvironment.Tofurtherclarifythisfinding,

weincubatedareducedAMCaseamount(0.1mU)with(GlcNAc)6

or 4NP-(GlcNAc)2, a chromogenicsubstrate used forevaluating chitinolyticpropertiesoftherecombinantenzyme,atpHranging from2.0to8.0at37◦Cfor1h

FACE analysis using pre-acidification step indicated that AMCase produced primarily (GlcNAc)2 and (GlcNAc)3 at pH 2.0∼5.0withasecondpeak(GlcNAc)2atpH6.0andseemedtobe activeuptopH8.0(Fig.6A).Thecolorimetricanalysisusing 4NP-(GlcNAc)2at405nmindicatedthatitscleavagereachedmaximum

atpH2.0anditdecreasedatmoreneutralpH(pH3.0∼7.0)(Fig.6B) Althoughbothanalysisshowedsimilarresults,FACEanalysiscould detectmorepronouncedsecondpeakatpH6.0(Fig.6C)

OurresultsindicatethatAMCasecandegradechitinsubstrates

to(GlcNAc)2atpH2.0∼8.0.ThesedatashowthattheFACE anal-ysiswithpre-acidificationismuchmoresensitivethantheassay usingchromogenicsubstrate,4NP-(GlcNAc)2.Ourresultssuggest thatAMCasecandegradechitinsubstratesnotonlyinstomachbut alsoinothertissuesincludinglungsandsubmaxillaryglands 3.7 DetectionofGlcNAcoligomersusingHPLCorFACE Next, using HPLC orFACE, we evaluated thedegradation of (GlcNAc)6byAMCaseatstrongacidicandneutralconditions.First,

wedegraded(GlcNAc)6 in McIlvaine’sbufferusingAMCaseand analyzedtheHPLC-separatedtheproductshavingabsorbanceat

210nm.We obtainedbuffer-derived peaks,whichare elutedat approximatepositionsof(GlcNAc)4∼6,whereasourimprovedFACE methoddetectedproper(GlcNAc) and(GlcNAc) signal

(Supple-Fig 1.Detection of pH-dependent fluorophore-labeled bands at pH 5.0 ∼ 8.0 Chitinolytic activity of mouse AMCase was investigated by incubating the enzyme (0.8 mU) with colloidal chitin or (GlcNAc) 6 at pH 2.0 ∼ 8.0 in McIlvaine’s buffer, followed by FACE as described in Materials and methods Chitin oligomers are shown in the left margin

as standards Recombinant AMCase degraded colloidal chitin (A) or (GlcNAc) 6 (B) and generated primarily (GlcNAc) 2 fragments, and in lower amounts the GlcNAc monomer and (GlcNAc) 3 The quantification is shown right Fluorescence intensity estimated from the results in Fig 1 A and B Thin arrows, (GlcNAc) 2 fragments; thick arrows, slow mobile bands.

Fig 2.Comparison of the fluorescent labeling between Jackson’s and our pre-acidification methods A Efficiencies of the Jackson’s method at pH 2.0 (left) or 7.0 (center) using McIlvaine’s buffer The quantification is shown right B The efficiency of the fluorescent labeling chitin oligosaccharide with pre-acidification at pH 2.0 (left) or 7.0 (center) The quantification is shown right.

Fig 3.Effect of different buffers on the labeling of the ANTS conjugate We tested for labeling of (GlcNAc)1∼6using McIlvaine’s buffer (A), phosphate or Tris-HCl buffer (B) and Good buffers (MES, PIPES, HEPES and MOPS) (C) The final concentration of each buffer was adjusted to 150 mM at pH 7.0 M, molecular weight markers of N-acetyl chitooligosaccharides; J, Jackson’s method; P, pre-acidification method.

Fig 4. Quantification of (GlcNAc) 2 using standard curve A To evaluate the

abil-ity for GlcNAc dimers quantification, we labeled (GlcNAc) 2 fragments at pH 7.0 B.

Quantification of GlcNAc oligomer was tested at up to 50 nmol of (GlcNAc) 2 There

was a linearity between (GlcNAc) 2 and fluorescence intensity in the whole range at

pH 7.0 The linearity was preserved when using pre-acidification.

mentaryFig.S1AandB).Thisisprobablycausedbythecitrateand

phosphateinMcIlvaine’sbufferhavingabsorbanceat210nm

Next,wechangedthebuffersystemusingGly-HCl(pH2.0)and Tris-HCl(pH7.0),respectively.Wefirstconfirmedthatthesebuffer systemsgavenosignificantbackgroundexceptforaminorpeak

atpH2.0ataroundexpected(GlcNAc)2signal(SupplementaryFig S2AandB)

Usingthesebuffersystems,weanalyzedthedegradedproducts

byHPLCandourimprovedFACEmethods.Theresultspresented

inFigs.5and6canbereplicatedbyHPLCatpH2.0andpH7.0, indicatingthat AMCaseproduces (GlcNAc)2 atstrongacidicand neutralconditions(Fig.7AandB).ForFACEanalysis,weuseda

10×dilutedsamplebecauseourimprovedmethodisvery sensi-tive(pmolamounts)whencomparedwiththeconventionalHPLC (Fig.7 andD).Basedontheseresults,weconcludethatAMCase generates(GlcNAc)2 underphysiologicalconditionsandthatour improvedFACEmethodisverysensitiveandeffectiveforthe detec-tionandquantificationofchitinoligomers

4 Discussion

AMCase has been shown to be predominantly produced in mousestomach(Bootetal.,2005;Ohnoetal.,2013;Ohnoetal.,

2012)and tohave maximal enzymatic activityatpH 2.0 (Boot

etal.,2001;Kashimuraetal.,2013).Previousstudieswerecarried outusingnaturalchitinsubstratesatacidicconditionsconcluding thatAMCaseproducesprimarily(GlcNAc)2atpH2.0(Bootetal., 2001;Kashimuraetal.,2015;Kashimuraetal.,2013).Ithasalso beenshownthatAMCasecanfunctionasadigestiveenzymeinthe mousegastrointestinaltract(Ohnoetal.,2016;Ohnoetal.,2013; Ohnoetal.,2012)

Fig 5. Re-examination of the pH degradation of chitin substrates by AMCase using pre-acidification method We performed same experiments in Fig 1 A and B except for labeling by ANTS using our pre-acidification method.

Fig 6.Chitinolytic properties of AMCase under somatic tissue pH conditions A The enzymatic reactions using (GlcNAc) 6 were carried out in a volume of 50 ␮L containing 0.1

mU E coli-expressed mouse AMCase in McIlvaine’s buffer B The pH dependence of chitinolytic activity of AMCase using 4NP-(GlcNAc) 2 as a substrate The values represent the percentage of the maximum activity at pH 2.0 C Comparison of the chitinolytic activity of recombinant AMCase analyzed by our improved FACE method ( Fig 6 A) and the colorimetric analysis using 4NP-(GlcNAc) 2 at 405 nm ( Fig 6 B) The values were represented as percentage of the maximum activity at pH 2.0.

OurpreviousgeneexpressionanalysisrevealedthatAMCase

mRNA is also expressed at high levels in salivary gland

and lung tissues (Ohno et al., 2012) Chitin oligomers

(N-acetyl-chitooligosaccharides)havevariousbiologicalactivitiesin

mammaliancells(Aametal.,2010;Khoushab&Yamabhai,2010)

Anti-cancerandanti-inflammatorypropertieshavebeenreported

forbothchitinandchitosanoligosaccharides(Azumaetal.,2015;

Masudaetal.,2014).MouseAMCasehasbeenshowntobeactive

atuptopH8.0,althoughitsactivitysubstantiallydecreaseswith higherpH(Bootetal.,2001;Kashimuraetal.,2013).We hypoth-esizedthatunderpathologicalconditions,AMCaseisupregulated andcangeneratespecificdegradationproductsdifferentfromthose

instomachconditionandthattheycanmodifythebioactivityofthe chitindegradationproducts.Therefore,wehavebeeninterestedin

Fig 7.Detection of GlcNAc oligomers using HPLC and FACE methods (GlcNAc) 6 was degraded by AMCase at pH 2.0 or pH 7.0 as described in Materials and Methods The samples were analyzed by HPLC (A) or FACE (B) Numbers with arrows indicate the number of GlcNAc units in the corresponding peaks For FACE, we used a 10 x diluted sample C Analysis of HPLC-separated degradation products by HPLC D Quantification of data generated by HPLC and FACE.

theAMCase-mediateddegradationpatternofchitinsubstratesat

neutralconditions

Thewidely-usedJacksonmethodforoligosaccharidesdetection

(Jackson,1990)isusedforreducingendslabelingintheanalyzed

moleculesfollowedbyhighresolutionPAGE.Weusuallyconduct

chitindegradation reactionusing McIlvaine’sbuffer, which is a

citrate/phosphatebuffersystemallowingustosetthepHovera

broadrange.However,wefoundtwodrawbacksofthismethodfor

chitinoligosaccharideslabelingdirectlyusedinhighconcentration

ofnon-volatilebuffer.ByFACEanalysis,wenoticedthatAMCase

producednotonlythepredominantGlcNAcdimerbutalsocertain

numberoffragmentswithslowermobilityatpH>5.0.Wefound

thattheseadditionalfragments(herecalled“by-products”)were

generatedatpH5.0∼8.0notduringthedigestionbyAMCasebut

duringtheoligomerslabeling(Fig.1).Importantly,weobserved

reducedlabelingathigherpHvalueswhencompared topH2.0

(Fig.2A)

To overcome these problems, we developed an improved

methodforfluorescentlabelingenablingustoefficientlylabelthe

reducingendsoftheGlcNAcoligomersgeneratedduringthe

enzy-maticreactionsperformedat pH2.0∼8.0 Weachievedthis by

acidificationofthesamplebyconcentratedaceticacidjustbefore

thelabelingstepwiththefluorophore.Importantly,during

label-ing,pHhastobeadjustedtopH<4.0.TheoriginalJacksonmethod,

however,issuitableforpH<4.0,adjustedbyadditionof

concen-tratedaceticacidtothedriedsample(Jackson,1990).Althoughwe

donotknowthechemicalstructureoftheby-products,our

pre-acidificationprocedurewasabletosuppressthesidereactionand

formsinglebandscorrespondingtotheGlcNAcoligomermarkers

(Fig.2B).Thismethodissimpleandcanbeappliedtovariousbuffers

commonlyusedforbiochemicalanalyses(Fig.3).Inaddition,the

fluorescenceefficiencyincreasedallowingbetterquantificationof

theGlcNAcoligomers.Ourpre-acidificationmethodpresentedin

thisreport[diagrammaticschemeofoverallourimprovedmethod

and the formation of ANTS-(GlcNAc)2 are described in

Supple-mentaryFigs.S3andS4]isessentiallyidenticalwiththemethods

reportedbyJackson(Jackson,1990)exceptforadditionof concen-tratedaceticacidbeforefluorescentlabelingreaction

IntheoriginalJacksonmethod(Jackson,1990),thesaccharide samplestobelabeledwereinitiallydissolvedinwaterorelse dis-solvedin0.1Mammoniumacetate,thendriedundervacuumand re-dissolvedinwater.Becauseammoniumacetateisvolatile,the driedsamplewouldbebuffer-freebeforere-dissolvinginwater Then,forthelabelingreactionthedilutedaceticacidissufficient

tomaintaintheacidicenvironmentneededtocatalyzethe open-ingofthereducingendsugar.But,inourexperiments,allsamples weredissolvedinhighconcentrationofnon-volatilebufferbefore labeling.Thisiswhywefounditnecessarytoaddmoreaceticacid,

inordertoensureanacidicsolutionduringthelabelingreaction Ourimprovedmethodprovidesasimplesolutiontothe observa-tionofunknownsideproductsinthefluorescentlabelingofchitin oligosaccharidesatthereducingend,foruseinFACEanalysisof chitinoligosaccharides.Itcouldbecomewidelyadoptedbyother laboratories

Finally,were-examinedthepH-dependentdegradationofthe chitinsubstratesusingourpre-acidificationmethodforlabelingthe GlcNAcoligomers(Figs.5and6).Wenoticedthat,atpH5∼8,the productsgeneratedfromchitinsubstratesaremoreabundantthan thosefromthesyntheticchromogenicsubstrateof4NP-(GlcNAc)2 (Fig.6)(Kashimuraetal.,2013).Thesedataindicatethatthe degra-dationabilityofAMCaseagainstcolloidalchitinissuperiortothe syntheticsubstratesatpH5∼8andthatAMCasecanworkunder somatictissuepHconditions.Family18chitinasesareproposedto utilizethesubstrate-assistedcatalyticmechanismwhiletheir cat-alyticdomainshavingTIM-barrelfold(TerwisschavanScheltinga

et al., 1995).The DXXDXDXE motif included in this domainis thoughttohaveacentralroleinsubstratebindingandcatalysis

inacidiccondition(Chouetal.,2006)withHis187ofAMCasebeing responsiblefortheacidicoptimum(Bussink,Vreede,Aerts,&Boot,

2008).Thisfoldingmechanismcouldexplainthemouse AMCase-mediated(GlcNAc)2productionatpH2.0.Themechanisticdetails

oftheAMCaseactivityatpH7.0remainstobedetermined.Taken

anddegradechitinsubstratestoproducepredominantly(GlcNAc)2

WedidnotobtainanyindicationoflongerGlcNAcoligomers

productionunderphysiologicalconditions.Onecanspeculatethat

AMCase-mediatedlongerGlcNAcoligomersproductionmayoccur

underpathologicalconditions.Ontheotherhand,longoligomers

produced byinefficient chitin degradation, mightbe stimuli or

enhancersofcertainpathologies

5 Conclusions

MouseAMCase hasoptimalpHat pH2.0 for themaximum

activityandisactiveatuptopH8.Weperformedanextensive

analysisofchitindegradationbyAMCasenotonlyinstrongacidic,

but alsoweak acidic toneutral conditions.When we analyzed

chitindegradationproductsinweakacidictoneutralconditionsby

FACEestablishedoriginallyforoligosaccharidesanalysis,wefound

thattheby-productsinthefluoresceinatedreactionwereformed

bylabelingatpH>5andestablisheda pre-acidificationmethod

forchitinoligosaccharideanalysistosuppresstheseproducts

for-mations.Usingthisprocedure,wefoundthatAMCasegenerates

(GlcNAc)2and(GlcNAc)3atconditionsmimickingsomatictissue

pHconditionsaswellasatpH2.0

Acknowledgements

WearegratefultoDaisukeYamanaka,NaohitoOhno,Tadatomo

Kawai,ShinjiNagumo,YasutadaImamuraandYoshiakiFurukawa

for their suggestions and encouragement, to Kazuaki Okawa,

DaisukeMizutani,EriTabataforvaluablesuggestions.Thiswork

wassupportedbytheProjectResearchGrantfromtheResearch

Institute of Science and Technology, Kogakuin University, and

Grants-in-Aidfor Scientific Research (15J10960 and 16K07699)

fromtheMinistryofEducation,Culture,Sports,Science,and

Tech-nologyofJapan,andGrantfromtheScienceResearchPromotion

Fundof thePromotion and Mutual Aid Corporationfor Private

SchoolsofJapanandinpartbyagrantoftheStrategicResearch

FoundationGrant-aidedProjectforPrivateUniversities(S1411005)

fromtheMinistryofEducation,Culture,Sport,Scienceand

Technol-ogy,Japan.P.O.B.receivedsupportfromALSAssociationandMayo

ClinicCenterforRegenerativeMedicine

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.carbpol.2017.01

095

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