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A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate

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Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even
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  Kazuyuki Sugahara and Fuchuan LiWenjun Han, Wenshuang Wang, Mei Zhao,  Chondroitin SulfateBacterium That Degrades Hyaluronan and A Novel Eliminase from a Marine Glycobiology and Extracellular Matrices: doi: 10.1074/jbc.M114.590752 srcinally published online August 13, 2014 2014, 289:27886-27898.J. Biol. Chem. 10.1074/jbc.M114.590752Access the most updated version of this article at doi: .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the  Alerts: When a correction for this article is posted• When this article is cited• to choose from all of JBC's e-mail alertsClick here   http://www.jbc.org/content/289/40/27886.full.html#ref-list-1This article cites 62 references, 38 of which can be accessed free at   a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  ANovelEliminasefromaMarineBacteriumThatDegradesHyaluronanandChondroitinSulfate * Receivedforpublication,June20,2014,andinrevisedform,August11,2014  Published,JBCPapersinPress,August13,2014,DOI10.1074/jbc.M114.590752 WenjunHan ‡§1 ,WenshuangWang ‡§1 ,MeiZhao ‡§ ,KazuyukiSugahara ¶ ,andFuchuanLi ‡§2 Fromthe ‡ NationalGlycoengineeringResearchCenter,and  § StateKeyLaboratoryofMicrobialTechnology,ShandongUniversity, Jinan250100,Chinaand  ¶ ProteoglycanSignalingandTherapeuticsResearchGroup,FacultyofAdvancedLifeScience,HokkaidoUniversityGraduateSchoolofLifeScience,Sapporo 001-0021, Japan Background:  Glycosaminoglycan (GAG) lyases have been widely isolated from terrestrial but not marine bacteria. Results:  A novel GAG lyase (HCLase) was identified for the first time from a marine bacterium. Conclusion:  The HCLase has very low homology to the characterized conventional GAG lyases and possesses very uniquebiochemical characteristics. Significance:  HCLase will be useful for chondroitin sulfate/hyaluronan-related research and applications. Lyases cleave glycosaminoglycans (GAGs) in an eliminativemechanismandareimportanttoolsforthestructuralanalysisandoligosaccharide preparation of GAGs. Various GAG lyases havebeen identified from terrestrial but not marine organisms eventhough marine animals are rich in GAGs with unique structuresand functions. Herein we isolated a novel GAG lyase for the firsttimefromthemarinebacterium Vibrio sp.FC509andthenrecom-binantly expressed and characterized it. It showed strong lyaseactivitytowardhyaluronan(HA)andchondroitinsulfate(CS)and wasdesignatedasHAandCSlyase(HCLase).Itexhibitedthehigh-estactivitiestobothsubstratesatpH8.0and0.5 M NaClat30°C.ItsactivitytowardHAwaslesssensitivetopHthanitsCSlyaseactivity.As withmostothermarineenzymes,HCLaseisahalophilicenzymeand verystableattemperaturesfrom0to40°Cforupto24h,butitsactivity isindependentofdivalentmetalions.ThespecificactivityofHCLaseagainstHAandCSreachedamarkedlyhighlevelofhundredsofthou-sandsunits/mgofproteinunderoptimumconditions.TheHCLase-resistant tetrasaccharide   4,5 HexUA  1-3GalNAc(6- O -sulfate)  1-4GlcUA(2- O -sulfate)  1-3GalNAc(6- O -sulfate) was isolated fromCS-D,thestructureofwhichindicatedthatHCLasecouldnotcleavethegalactosaminidiclinkageboundto2- O -sulfated D -glucuronicacid(GlcUA) in CS chains. Site-directed mutagenesis indicated thatHCLasemayworkviaacatalyticmechanisminwhichTyr-Hisactsasthe Brønsted base and acid. Thus, the identification of HCLase pro- videsausefultoolforHA-andCS-relatedresearchandapplications. Chondroitin sulfate (CS), 3 synthesized as the glycosamin-oglycan (GAG) side chains of proteoglycans (1), is widely expressedoncellsurfacesandinextracellularmatricesandpar-ticipates in various biological events, including development of the central nervous system (2, 3), wound repair (4, 5), viralattachment(6–8),growthfactorsignaling(9,10),morphogen-esis (11), and cytokinesis (12–14). The various functions of CShave been attributed to their structural diversity. CS chains arecomposedofrepeatingdisaccharideunitsofGlcUA-GalNAcinwhich GlcUA and GalNAc represent  D -glucuronic acid and  N  -acetyl- D -galactosamine, respectively. In biosynthesis, afteror while CS chains are polymerized by various enzyme com-plexes, each of which is formed by heterologous combinationsoftwoofthesixchondroitinsynthasefamilymembers(15–17),CS chains are further modified by differential sulfation by spe-cific sulfotransferases at C-2 of GlcUA/ L -iduronic acid and/orC-4 and/or C-6 of GalNAc to yield prominent structural diver-sity (18). Furthermore, some GlcUA residues are epimerizedinto  L -iduronic acid (IdoUA) by the action of glucuronyl C-5epimerase, and the chain that contains repeating disaccharideunitsof-IdoUA-GalNAc-hasbeendesignatedasdermatansul-fate (DS) (19, 20). Therefore, CS and DS chains are oftendetected as co-polymeric structures (CS-DS) and are morelikely to be periodically distributed in cell/tissue-specific man-ners (14, 21). The introduction of these unique structuraldomains into CS-DS chains in respective tissues results in dif-ferent responses to various CS- or DS-binding proteins such asheparin-binding growth factors and cytokines (22).Prominent structural heterogeneity has hampered detailedstructural and functional analyses of CS and DS chains. Spec-troscopic techniques such as nuclear magnetic resonance(NMR) and mass spectrometry (MS) are widely used to struc-turally determine CS/DS oligosaccharides (23). Furthermore,some CS/DS-degrading enzymes, particularly bacterial CS/DS *  ThisworkwassupportedbyNationalHighTechnologyResearchandDevel-opment Program of China Grant 2012AA021504, Major State BasicResearch Development Program of China Grant 2012CB822102, Shan-dong Province Science and Technology Development Plan Grant2013GSF12106, General Financial Grant from China Postdoctoral ScienceFoundation Grant 2013M531588, and Specialized Research Fund for theDoctoralProgramofHigherEducationGrant20130131120079andinpartby Grant-in-aid for Challenging Exploratory Research 25670018 from theJapan Society for the Promotion of Science. The nucleotide sequence(s) reported in this paper has been submitted to theGenBank   TM  /EBI Data Bank with accession number(s) KJ885185. 1 Both authors contributed equally to this work. 2  Towhomcorrespondenceshouldbeaddressed:NationalGlycoengineeringResearch Center, Shandong University, 27 S. Shanda Rd., Jinan 250100,China. Tel.: 86-531-88365165; Fax: 86-531-88363002; E-mail: fuchuanli@sdu.edu.cn. 3  The abbreviations used are: CS, chondroitin sulfate; DS, dermatan sulfate;HA, hyaluronan; GAG, glycosaminoglycan; GlcUA,  D -glucuronic acid;HexUA, hexuronic acid;  4,5 HexUA,  4,5 -unsaturated hexuronic acid; 2S,2- O -sulfate; 6S, 6- O -sulfate; CSase, chondroitinase; 2-AB, 2-aminobenz-amide;IdoUA, L -iduronicacid;BM,basalmedium;HCLase,HAandCSlyase;rHCLase, recombinant HCLase; Ni-NTA, nickel-nitrilotriacetic acid.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 40, pp. 27886–27898, October 3, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 27886  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 289•NUMBER 40• OCTOBER3,2014   a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  lyases with specific activities, are being used in the structuraland functional analyses of CS/DS chains in conjunction withspectroscopic techniques (23–25). CS/DS lyases depolymerizeCS/DS via a   -elimination reaction, which cleaves the galac-tosaminidic linkages to uronic acid residues to yield an unsat-urated 4,5-bond between C-4 and C-5 on the uronic acid resi-due at the site of cleavage (26). Several CS/DS lyases are now commercially available, including chondroitinase (CSase) ABCfrom  Proteusvulgaris (27),whichdigestsbothCSandDSaswellas hyaluronan (HA). CSase ACI from  Flavobacterium hepari-num  (27) and CSase ACII from  Arthrobacter aurescens  (28)bothspecificallycleaveCS,whereasCSaseBfrom  F.heparinum only cleaves the galactosaminidic linkages attached to theIdoUA residues of DS as well as CS-DS hetero- and polymers.CS/DS lyases are not only useful tools for investigating thestructure-function relationship and the preparation of bioac-tive oligosaccharides of CS/DS but are also potential therapeu-tic agents for the treatment of injuries to the central nervoussystem (29). Therefore, screening and identifying novel CS/DSlyases with high activity and specificity are important for bothacademic research and applications.The ocean is regarded as the srcin of life on Earth and hostsmost life forms. The unique marine environment as a powerfulselective force prompts marine organisms to generate specificandpotentbioactivemoleculestoadapttocomplicatedmarineenvironments. Marine animals are immense sources of GAGswithuniquestructuresandbioactivities.Threeofthefivecom-mercially available CS/DS subtypes, namely CS-C, CS-D, andCS-E, are derived from shark cartilage, shark fins, and squidcartilage, respectively. In addition, we and others have purified variousnovelCS,DS,andCS-DSpreparationsfromseaanimals(30–34). The abundant presence of these unique structures inmarineenvironmentsindicatestheexistenceofCS/DS-degrad-ing microbes. Therefore, marine microbes may be an idealsource for identifying novel CS/DS lyases with extraordinary properties. However, to the best of our knowledge, no CS/DSlyase from marine bacteria has yet been characterized in detail.In the present study, a marine  Vibrio  strain with high GAG-degrading capacity was isolated using CS-C from shark carti-lage as the sole carbon source. The bacterial genome wassequenced to identify the genes of GAG-degrading enzymes. Aputative GAG lyase gene (HA and CS lyase ( hclase )) wasexpressed in  Escherichia coli , and the recombinant HCLaseprotein was purified to analyze sequence properties, substratespectra, and enzymatic characteristics. An HCLase-resistantCS tetrasaccharide was isolated from CS-D and sequenced by enzymaticdigestionfollowedbyHPLCanalysis.Thekeyaminoacid residues at the active site of HCLase were identified by site-directed mutagenesis. Our results demonstrated thatHCLase exhibited very high activity and specificity for thedigestion of CS and HA and will be a useful tool for basicresearch on and applications of CS and HA. EXPERIMENTALPROCEDURES  Materials— The strains and plasmids used in this study arelisted in Table 1. SDS, proteinase K, PrimeSTAR TM HS DNApolymerases, restriction endonuclease, and other genetic engi-neering enzymes were purchased from Takara Inc. (Dalian,China). Standard unsaturated disaccharides, chondroitin,CS-A from whale cartilage, CS-C and CS-D from shark carti-lage, CS-E from squid cartilage, and DS from porcine skin werepurchasedfromSeikagakuCorp.(Tokyo,Japan).2-Aminoben-zamide (2-AB), cyanoborohydride (NaBH 3 CN), CSase ABC(EC 4.2.2.20), heparin, heparan sulfate, alginate, and xanthanwereobtainedbySigma.Allotherchemicalsandreagentswereof the highest quality available. CS or HA tetra-, hexa-, andoctasaccharideswerepreparedbythedigestionofCS-AorHA,respectively, using CSase ABC followed by gel filtration chro-matography on a Superdex TM Peptide 10/300 GL column asdescribed previously (24). TABLE1 Bacterialstrains,plasmids,andprimersusedforsequencinginthepresentstudy Restriction enzyme sites are underlined. Ap r , ampicillin-resistant; Kan r , kanamycin-resistant. Strains and plasmids Description SourceStrains Vibrio  sp. FC509 A chondroitin sulfate-degrading marine bacterium that secretes multiple GAG lyases (patented asCGMCC 8913)This study   E. coli  BL21(DE3)  F   ,  ompT  ,  hsdSB  ( rB  ,  mB  ),  dcm ,  gal  ,  (  DE3 ),  pLysS  ,  Cm r  Novagen  E. coli  Top10  F   mcrA  ( mrr-hsdRMS-mcrBC  )   80lacZ    M15  lacX74 deoR recA1 araD139  ( araA-leu ) 7697  galU galK rpsL endA1 nupG  Invitrogen Plasmids pBAD/gIII A Expression vector; Ap r InvitrogenpCold TF Expression vector; Ap r TakarapET22b Expression vector; Ap r NovagenpET30a Expression vector; Kan r NovagenpET22b-HCLase pET22b carrying an amplified NcoI-XhoI fragment encoding the recombinant protein of HCLasefused with a His 6  tag at the C terminusThis study  Sequencing primers HCLase-F 5  -GCCATGGATATGCGATGACCACCAGTTCACTG-3  HCLase-R 5  -GCTCGAGTCGCACTGAAAATTGATAACTTTGTCC-3  HCLase-W492A-F 5  -CCCGTGGTCGATAACCATCGACTCGC-3  HCLase-W492A-R 5  -TGCGTAATCGGTGTAGTGGTCTTGCTGGC-3  HCLase-H285A-F 5  -GACATTGCTTATAACGGCACTTATGGC-3  HCLase-H285A-R 5  -GCCTGCCTGAATAAAGGAGCCATCTTGG-3  HCLase-Y290A-F 5  -AACCGGCACTTATGGCAACGTGCTACTGGG-3  HCLase-Y290A-R 5  -TGCAGCAATGTCGCCATGCTGAATAAAGGAG-3  HCLase-Y294A-F 5  -GGCAACGTGCTACTGGGTGGGCTTGGC-3  HCLase-Y294A-R 5  -TGCAGTGCCGTTATAAGCAATGTCGCATGCTG-3   ANovelGlycosaminoglycanEliminasefromaMarineBacterium OCTOBER3,2014• VOLUME 289•NUMBER 40  JOURNAL OF BIOLOGICAL CHEMISTRY   27887   a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   Identification of Marine CS-degrading Bacteria— Coastalsediments were collected from Jiaozhou Bay (N36°3  39  –N36°3  43  , E120°18  31  –E120°19  15  ) nearby Qingdao City inShandong Province, China. Basal medium (BM) composed of 3.0% (w/v) NaCl, 0.3% (w/v) KH 2 PO 4 , 0.7% (w/v) K 2 HPO 4  3H 2 O, 0.2% (w/v) (NH 4 ) 2 SO 4 , 0.01% (w/v) MgSO 4 , 0.01% (w/v)FeSO 4  7H 2 O,0.05%(w/v)chondroitinsulfatefromsharkcarti-lage (CS-C), and 1.5% (w/v) agar (pH 7.2) was used to isolateCS-degrading bacteria from the sediments. After being incu-bated at 30 °C for 72 h, colonies on the BM were randomly selected and transferred to fresh plates for further purification.To assay the polysaccharide-degrading abilities of the isolates,BM broth without agar was supplemented with various poly-saccharides (alginate, chondroitin sulfates C, dermatan sulfate,heparin, heparin sulfate, hyaluronate, and xanthan) as the solecarbon source at a final concentration of 0.5% (w/v). Bacterialgrowth was evaluated by measuring absorbance at 600 nm(  A 600 ).The genomic DNA of individual CS-degrading bacteria wasprepared using SDS and a proteinase K treatment. PCR ampli-fication of the 16 S rRNA gene sequence was performed usingthe bacterial universal primer pair 27f (5  -GAGTTTGATC-CTGGCTCAG-3  ) and 1492r (5  -AAGGAGGTGATCCA-GCC-3  ) (35). Gel-recovered PCR products were cloned intothe pMD19-T vector (Takara Inc.) for sequencing. Thesequence was analyzed against the GenBank TM database usingthe on-line BLAST program to search for the most similarsequences. A phylogenetic tree was generated using the neigh-bor joining method of Nei and co-workers (36) with MEGA version 5.05. CloningofGAGLyaseGenes— Thedraftgenomeof  Vibrio sp.FC509 was sequenced at Meiji Biotech Inc. (Shanghai, China)using Roche Applied Science 454 GsFLX, IlluminaGAIIxtech-nology.Thesequenceof  Vibrio sp.FC509wasannotatedatOakRidge National Laboratory using their genome annotationpipeline. This included the application of a number of annota-tion programs beginning with open reading frame (ORF) pre-diction using Prodigal (37) followed by manual annotationusing the JGI GenePRIMP pipeline (38). Automated proteinfunction prediction was then performed using a number of databases, including protein domains (Pfam), UniProt (39),TIGRFAMs (40), KEGG (41), InterPro (42), and COG (43);metabolic reconstruction analysis using PRIAM (44); signalpeptide prediction using SignalP (45); tRNA prediction usingtRNAscan-SE(46);andrRNApredictionusingRNAmmer(47). Sequence Analyses of Genes and Proteins of Chondroitin Lyases— Promoter motifs of the 5  -flanking DNA regionupstreamoftheORFwereidentifiedusingPrimerPremierver-sion 5.0 (PREMIER Biosoft International, Palo Alto, CA) andthe Promoter 2.0 Prediction Server. The GC content (G  C%)of the ORF was calculated using Bio-Edit version 7.0.5.3.A similarity search of the protein sequence was performedusing the BLASTp algorithm online. Secretion signal peptidesand their types were identified using the SignalP 4.0 server andLipoP 1.0 server, respectively. The molecular mass of the pro-tein was estimated using the peptide mass tool on the ExPASy server of the Swiss Institute of Bioinformatics. Sequence align-ment and phylogenetic analysis were performed using MEGA version 5.05. Protein modules and domains were identifiedusing the Simple Modular Architecture Research Tool, Pfamdatabase, and Carbohydrate-Active Enzyme database.  Heterologous Expression of the HCLase Gene— To expressHCLase in  E. coli  strains, the full-length gene of HCLase wasamplified using primer pairs (as listed in Table 1) and highfidelity PrimeSTAR HS DNA polymerases (Takara Inc.).Primer pairs with restriction enzyme sites (underlined) weredesigned according to the inserting site sequences of theexpressionplasmids,includingpBAD/gIIIA(Invitrogen),pET-22b(  ), pET-30a(  ) (Novagen), and pCold TF (Takara Inc.).Gel-recoveredPCRproductswereclonedintotheseexpression vectors. The expression plasmid (pBA-HCLase) constructedfrom pBAD/gIII A was transformed into  E. coli  Top10 cells,whereas another expression vector from pET-22b(  ) (pE22b-rHCLase), pET-30a(  ) (pE30a-rHCLase), or pCold TF (pCTF-rHCLase) was transformed into  E. coli  BL21(DE3) cells. Theintegrity of the nucleotide sequences of all constructed plas-mids was confirmed via DNA sequencing.  E. coli cells harboring an expression vector were initially cul-tured in LB broth. When cell density reached an  A 600  of 0.8–1.0, the broth was supplemented with the inducer ( L -arabinoseor isopropyl 1-thio-  - D -galactopyranoside) at a final concen-tration of 0.01 m M  to start the expression of targeting proteins.Afteracontinualcultivationforanadditional24hat16 °C,cellswere harvested by centrifugation at 6,000    g   for 15 min,washedtwiceusingice-coldbufferA(50m M Tris-HCl,150m M NaCl (pH 8.0)), resuspended in buffer A, and disrupted by son-ication (50 repetitions, 5 s) in an ice-cold environment. Aftercentrifugation at 15,000   g   for 30 min, the supernatant wascollected for further purification of soluble targeting proteins.  PurificationofRecombinantProteinrHCLase— TopurifytherHCLaseprotein,thesupernatantcontainingthesolublenativeenzyme was loaded onto a column packed with nickel-Sephar-ose TM 6 Fast Flow (GE Healthcare), then the column waswashed with buffer A containing 50 m M  imidazole to removeimpurities, and rHCLase was finally eluted from the Ni-NTAcolumn using a gradient concentration of imidazole, rangingfrom 50 to 250 m M . The purity of rHCLase was analyzed usingSDS-PAGE. SDS-PAGE was performed using 13.2% polyacryl-amide gels according to Sambrook  et al.  (48). Coomassie Bril-liant Blue R-250 was used to stain proteins in gels. Protein con-centrations were determined by the Folin-Lowry method (49).  Assay of rHCLase Activity toward Various PolysaccharideSubstrates— To determine the substrates of rHCLase, variouspolysaccharides ( e.g.  alginate, CS-A, CS-C, CS-D, CS-E, DS,heparin, heparin sulfate, hyaluronan, and xanthan) were indi- viduallydissolvedindeionizedwatertopreparestocksolutions(10mg/ml).Eachstocksolution(10  l)wasmixedwith20  lof 250 m M  NaH 2 PO 4 -Na 2 HPO 4  buffer (pH 7.0), 60   l of water,and 10   l of the appropriately diluted enzyme and then incu-batedat37 °Cfor12h.Enzyme-treatedpolysaccharidesampleswere heated in boiling water for 10 min and then cooled inice-coldwaterfor10min.Afterbeingcentrifugedat15,000   g  for 15 min, the supernatant was collected and analyzed by theabsorbance at 232 nm (27) and gel filtration HPLC.  ANovelGlycosaminoglycanEliminasefromaMarineBacterium 27888  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 289•NUMBER 40• OCTOBER3,2014   a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   Biochemical Characterization of the Recombinant ProteinrHCLase— To determine the optimal pH for rHCLase activity,HA and CS-C (1 mg/ml) were digested, respectively, withrHCLase (2 ng) in buffers with different pH values, including afinal concentration of 50 m M  NaAc-HAc buffer (pH 5.0–6.0),50 m M  NaH 2 PO 4 -Na 2 HPO 4  buffer (pH 6.0–8.0), and 50 m M Tris-HCl buffer (pH 7.0–10.0) in a total volume of 100   l, at30 °C for 10 min. After the optimum pH was determined, theeffects of temperature on rHCLase activity were tested in 50m M  NaH 2 PO 4 -Na 2 HPO 4  (pH 8.0) at temperatures from 0 to90 °C for 10 min. The effects of metal ions/chelating reagent (5m M ) and concentrations of NaCl (0–1  M ) on the HA- and CS-degrading activities of rHCLase were investigated at the opti-mum pH and temperature described above. To determine thethermostability of rHCLase, the enzyme in 50 m M  NaH 2 PO 4 -Na 2 HPO 4  buffer (pH 8.0) containing 0.5  M  NaCl was preincu-bated for 0–24 h at a temperature from 0 to 90 °C, and theresidualHA/CS-degradingactivitywasdeterminedat30 °C.Allreactions were performed in triplicate, and after each treat-ment, the activity of enzyme was estimated by measuring theabsorbance at 232 nm (27). Optimum Assay Conditions of rHCLase— The activities of rHCLase were measured according to the method provided by Yamagata  et al.  (27). Briefly, rHCLase (2 ng) was added to 1mg/ml GAGs in 50 m M  NaH 2 PO 4 -Na 2 HPO 4 , 500 m M  NaClbuffer (pH 8.0) in a total volume of 1 ml. The reaction mixturewasincubatedat30 °C.Atvarioustimeintervals(upto10min),aliquots of 100   l were withdrawn in duplicate, boiled for 10min, and then cooled in ice-cold water for 10 min. After beingcentrifuged at 15,000   g   for 15 min, the supernatant was col-lected, diluted five times, and analyzed by absorbance at 232nm. One unit of enzyme was defined as the amount of enzymethat produced 1  mol of unsaturated carbon bonds/min. Gel Filtration Chromatography— Samples digested withrHCLase were analyzed by gel filtration chromatography on aSuperdex Peptide 10/300 GL column. The mobile phase was0.20  M  NH 4 HCO 3  at a flow rate of 0.4 ml/min, and the elutedfractions were monitored at 232 nm using a UV detector.Onlinemonitoringanddataanalysis( e.g. molarrationdetermi-nation) were performed using the software LCsolution version1.25.  Digestion Pattern of Polysaccharides by rHCLase— To deter-minethedegradationpatternofrHCLase,thedigestsofHAandCS (1 mg/ml) by rHCLase (1 unit/ml) were traced at 30 °C.Aliquotsofthedegradationproducts(10  g)wereremovedfortime course experiments to analyze gel filtration patterns by monitoring at 232 nm.To further determine the molecular weights and struc-tural characteristics of the oligosaccharide products, 1 ml of  various polysaccharides (1 mg/ml) was digested usingrHCLase (1 unit/ml) at 30 °C for 10 min. The reaction mix-ture was heated in boiling water for 10 min and subsequently cooledto4 °C.Afterbeingcentrifugedat15,000   g  for30min,the supernatant was loaded onto a pre-equilibrated SuperdexPeptide 10/300 GL column. Fractionated oligosaccharide sam-pleswerecollectedforpuremonomersbyonlinemonitoringat232 nm and freeze-dried repeatedly to remove NH 4 HCO 3  forfurther identification.The major disaccharide fractions purified from HA andCS-A were further identified by electrospray ionization MS onan ion trap TOF hybrid mass spectrometer (LCMS-IT-TOF,Shimadzu, Japan). Electrospray ionization MS analysis was setin the negative ion mode and with the following parameters:source voltage at 3.6 kV, nebulizer nitrogen gas flow rate at 1.5liter/min, heat block and curved desolvation line temperatureat 200 °C, and detector voltage at 1.8 kV. The mass acquisitionrange was set at 200–600.  IsolationandSequenceofanrHCLase-resistantTetrasaccha-ride from CS-D— To prepare rHCLase-resistant tetrasaccha-rides,200  lofCS-D(1mg/ml)wasexhaustivelydigestedusingrHCLase (100 units/ml) at 30 °C for 72 h. The reaction mixturewasheatedinboilingwaterfor10minandsubsequentlycooledto 4 °C. After being centrifuged at 15,000   g   for 30 min, thesupernatant was loaded onto a pre-equilibrated Superdex Pep-tide 10/300 GL column. The tetrasaccharide fraction was col-lected by online monitoring at 232 nm and desalted by repeating freeze-drying. The tetrasaccharide fraction wassubfractionated by anion-exchange HPLC on a YMC-PackPolyamineIIcolumn(YMC,Kyoto,Japan).Thetetrasaccharidefraction was loaded on the column equilibrated with 16 m M NaH 2 PO 4 andthenelutedwithalineargradientfrom16to460m M  NaH 2 PO 4  over 60 min at a flow rate of 1.0 ml/min at roomtemperature. The eluates were monitored by measuringabsorbance at 232 nm, and a major peak was collected anddesalted with a Superdex Peptide column as described above.The purified HCLase-resistant tetrasaccharide was se-quencedatalowpicomolelevelbyCSasedigestioninconjunc-tion with HPLC. Briefly, to identify the composition of thedisaccharide,analiquot(5pmol)ofthetetrasaccharidefractionwas digested with CSase ABC (Sigma) and labeled with 2-ABand sodium cyanoborohydride reagents as described by Bigge et al.  (50). Free 2-AB was removed by extraction with chloro-form.Thetetrasaccharidefraction(30pmol)usedforsequenc-ing was labeled with 2-AB and purified by paper chromatogra-phy, and an aliquot (5 pmol) of 2-AB-labeled tetrasaccharidesamplewasthentreatedwithanovelexo-CSasefrom Vibrio sp.FC509. 4 All these preparations were individually analyzed by anion-exchange HPLC on a YMC-Pack Polyamine II columneluted with a linear gradient from 16 to 460 m M  NaH 2 PO 4  overa 60-min period and monitored using a fluorescence detector.  Effects of 2-AB Labeling on the Digestion of Oligosaccharidesby rHCLase— To investigate the effects of 2AB labeling on thedigestion of oligosaccharides by rHCLase, pure HA/CS oligo-saccharide monomers were fluorescently labeled with 2-ABand digested by rHCLase. The resulting digests (5-pmol sam-ples) were analyzed by gel filtration on a Superdex Peptide10/300GLcolumn(GEhealthcare),andthesamplesweremon-itored using a fluorescence detector with excitation and emis-sion wavelengths of 330 and 420 nm, respectively (51).  Expression and Characterization of Mutant rHCLase— Toinvestigate the functions of the specific amino acid residues inthe HA/CS-degrading activities of HCLase, the protein se-quence of HCLase was directly submitted to the SWISS- 4 W. Wang, W. Han, M. Zhao, and F. Li, unpublished data.  ANovelGlycosaminoglycanEliminasefromaMarineBacterium OCTOBER3,2014• VOLUME 289•NUMBER 40  JOURNAL OF BIOLOGICAL CHEMISTRY   27889   a  t   Uni   v e r  s i   t   y of  H a  w a i  i   a  t  M a n o a L i   b r  a r  y on O c  t   o b  e r  3  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om

assignment8 group5

Jan 20, 2019
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