非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究

物工程学报2005年1月Chinese Journal of Biotechnology非解朊梄热菌HG102耐热β-糖苷酶的结构与功能研究The Structure-function Relationship of Thermostable B-glycosidase from the Thermophilic Eubacterium Thermusnonproteolyticus HG102杨雪鹛2,杨寿钧,韩北忠2,金城YANG Xue-Peng. 2, YANG Shou-Jun, HAN Bei-Zhong and JIN Cheng1.中国科学院微生物研究所微生物资源前期开发国家重点实验室,北京1000802.中国农业大学食品科学与营养工程学院,北京1000831. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beying 100080, China2. College of Food Science and Nutritional Engineering, Beying 100083, China摘要非解朊栖热莤HGl0耐热β糖苷酶为(βα)桶状结构,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较,推测Chul64和Glu38分别是质子供体和亲核基团两个活性位点;在α-螺旋N端第一位的脯氡酸和蛋白质外周的精氣酸是耐热机制的关键位点和关键氯基酸残基。为确定这些氨基酸残基的功能,通过基因定点突变的方法分别把Gul64、Glu338Pmo316、Pm356、Pm344和Arg325置换成Cln、Ala、、Ala、Phe和Leu,同时还对Pm316和Pm356进行了双置换。突变酶经过纯化得到电泳纯,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析,结果表明Gul64和Gu38分别是质子供体和亲核基团,亲核基国的突变酶 TnglyE38A可以合威混合型糖苷健寡糖类似物;在α螺旋N端第一位的Pmo316和Pmo356以及在蛋白质外周形成离子键的Arg325均是对耐热性有贡献的关键氨基酸残基关键词β-糖苷酶,转糖苷活性,热稳定性,定点突变中图分类号Q814文献标识码A文章编号10003061(2005)01-008408Abstract B-Glycosidase(Tngly)from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostablemonomeric protein and adopts the(pla) barrel fold, is an excellent model system to be investigated for the thermostable mecha-nism,activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly( Wang et al2003)and comparison of other proteins structure of homofamily, Glul64 and Glu338 may act as proton donor and nucleophile inthe hydrolysis reaction respectively; proline located at NI of a-helix and arginine which can form ion link may contribute to thethermostability. We aim to further identify the critical sites and the amino acid residue(s)responsible for the activity, the thermal stability and the substrate specificity. Mutations had been constructed by site-directed mutagenesis. They are Glu164GInGlu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified toSDS-PAGE purity. Changes in the conformations were examined by means of CD. The Glu338 Ala mutant showed no detectablehydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. TheReceived: June 17, 2004; Accepted: July 26, 2004中国煤化工CNMHGw Corresponding author. Tel: 86-10-62587206: E-mail: jinc@ sun. imaccn中国科学院知识创新工程项目基金资助(No.0103)。杨雪鹏等:非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究Glul64 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-typewere determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at NI positions of a-helix, and Arg325 that form salt bridge between a-helices 5 and a-helices 6, are the critical sites toprotein thermostabilizaticKey words B-glycosidase, transglycosylation, thermostability, site-directed mutagenesisβ-糖苷酶(E.C.3.2.1.21)生物来源广泛,可以Cu38来鉴定P糖苷酶Tngy水解反应时的质子供水解多种B构型的糖苷键具有广阔的应用前景。体和亲核基团;置换Pm316、Arg325、Po344和Pm356嗜热细菌 Thermus nonproteolyticus HGI02的β糖苷酶来探讨酶的耐热分子基础。Tngy基因已克隆、表达并进行了酶学性质的研究2。Tngy属于糖苷酶家族1,具有葡萄糖苷酶、半1材料和方法乳糖苷酶、岩藻糖苷酶和甘露糖苷酶活性,在高温下1.1材料还具有转糖苷活性,它的最适水解反应温度和pH1.1.1菌株与质粒:大肠杆菌(E.col)AS1.1739值分别为90℃和56,在90℃时,酶的半衰期为25K12r△( lacIPOZY)×74]在中国科学院微生物研h。Tngy晶体结构解析表明,该酶为(β/a)桶状结究所购买,重组质粒pHY(在pUCl8载体HindⅢ克构,分别位于第四β-shet和第七β-shet上的Glu164隆位点含有β糖苷酶目的基因)为本实验室构建。和Gu338,可能为水解反应时的质子供体和亲核基BMH1-18购自 Promega公司。11.2培养基及培养条件:LB培养基为大肠杆菌B糖苷酶可用来合成寡糖。,但反应要在有机完全培养基,固体培养基加入1.5%的琼脂粉。重相中并需要高浓度的糖基供体。 Mackenzie等人将组菌培养时加入氨苄青霉素(100pg/mL),培养温度Agrobacterium sp.β葡萄糖苷酶的亲核基团谷氨酸为37℃;诱导时液体培养基加入1%(W/V)乳糖固残基突变成甘氨酸,使得突变的酶只能合成糖苷键,体培养基涂布4μLITG(200mg/mL)40 L X-gal(20不再具有水解功能,从而能使寡糖的产率达到mg/mL)。90%。B糖苷酶Tngy可以在65℃下水解乳糖或11.3酶及生化试剂:定点突变试剂盒 Geneen纤维二糖生成三糖2),说明酶的活性中心适合转糖 tor in vitro Site-Directed Mutagenesis System Kit购自苷反应,可用来合成寡糖,置换耐热β-糖苷酶的亲核 Promega公司;T4 Polynucleotide Kinase购自 Promega基团来合成寡糖更具有优势。已知结构的蛋白质大公司;溶菌酶购自华美公司;CMP3 Fluoro-neuram约10%为(pa)桶状结构,或叫TM结构,因此以Acid购自 Calbiochem-Novabiochem公司;胰化蛋白胨Tngy作为模型来研究(a桶状结构的稳定机制非( TRYPTONE)和酵母提取物( YEAST EXTRACT)购自常有意义。文献报道,在 a-helix N端第一位脯氨酸 OXOID公司;PTG、Xgl,ONPG和乳糖购自Sgma公的刚性结构和蛋白质外周精氨酸形成的离子键可能司;蛋白质分子量标准购自华美公司;其它试剂均为对蛋白质热稳定性有一定的贡献3分析纯试剂。本实验在Tngy晶体结构的基础上,用基因定所用诱变寡核苷酸为上海生工公司合成,见点突变的方法置换Tngy的氨基酸残基Gn164和表1。表1置换氨基酸位点及其在蛋白质二级结构中的位置和相应的诱变寡核苷酸设计Table 1 Oligonucleotides and mutagenic position in proteinGlu164GlnNo, 4, B-sheet5’- ACCCTGAACCAGCCCTGGTGO3′Glu338AlaNo. 7, B-sheet5.TACATCACGGCAAACGGGGCC-35'-GGGAGGTCTACGGCGAGGGGCTT-3lon linkNo.7,β-shee中国煤化工3No. 7. g-helix NICNMHG5'-GTGGAGGACGCCGACCGGGTG-3old and underlined nucleotides are the mutations sites86Chinese Journal of Biotechnology生物工程学报2005,vol.21,No.11.2方法SWISS- MODEL. htm.网站上完成801.2.1基因定点突变:以重组质粒pHY单链DNA12.6转糖苷反应:突变酶 TnglyE338A和各种底物为模板在诱变寡核苷酸介导下,用定点突变试剂盒在65℃,pH6.8下反应2h,薄层层析检测。相应的试剂进行突变和筛选。筛选出单菌株提取质1.27薄层层析(mLC):展开剂为正丁醇:乙酸:水粒送交 TaKaRa Biotechnology( Dalian)测序鉴定突变=1:2:1;显色剂为苯胺:二苯胺:磷酸=5:5:1(显色结果。范围为10g)。122突变基因的表达和蛋白质纯化:用突变的在数据的测定当中都进行3次或3次以上试重组质粒转化到大肠杆菌(E.coli)AS1.1739,从过验,误差范围在5%以内夜培养的 Amp-LB平皿上挑取单菌落,接种到5mL液体LB(Amp100g/mL)培养12h,1%接种量接种结果到100 mL Amp-LB液体培养基中培养12h;再以1%2.1基因定点突变构建突变酶基因的接种量接种到4 L Amp-LB液体培养基(5L发酵以单链重组质粒为模板,由诱变寡核苷酸和选罐)中培养,加入1%的乳糖诱导,37℃,300rmin的择寡核苷酸介导突变和筛选突变基因)。突变质搅拌速度,通无菌空气,培养28h粒与野生质粒大小相同。测序结果显示突变质粒收集发酵罐中的4L发酵液(4℃,6000g,15 pHYL64编码164位上的clu碱基密码子GAG突min)离心,菌体用磷酸缓冲液(50mml/L,pH66)悬变为cn的密码子CAG;突变质粒pHYE338A编码浮;冰浴超声破碎,离心去除细胞碎片;在80℃水浴338位上的Gu碱基密码子GAA突变为Ala的密码恒温加热15mn,离心(4℃,1000g,15min)取上清。子GCA;突变质粒pHYP36G编码316位上的Pro碱向上清液中缓慢加入固体硫酸铵,收集30%~60%基密码子CCC突变为Gy的密码子GGC;突变质粒饱和度的沉淀,溶于磷酸缓冲液中(50mmoL,pHYP356A编码356位上的Pro碱基密码子CCC突pH66),用同种缓冲液透析过夜。除盐的粗酶冻干变为Ala的密码子GCC;突变质粒pHYP344F编码浓缩后,在 AKTA FPLC蛋白质纯化系统上用DEAE344位上的Pmo碱基密码子CCC突变为Phe的密码离子交换柱进行纯化,缓冲液A为磷酸缓冲液(50子CTr;双突变质粒pHYP316G/P356A编码316位和mmol/L,pH6.6),缓冲液B为1mol/LNaC溶于磷酸356位上的Pmo碱基密码子CCC分别突变为Cy的缓冲液(50mmol/L,pH6.6),洗脱条件为在5个柱体密码子GCC和Ala的密码子GCC;突变质粒积内B溶液比例上升到30%,测酶活和 SDS-PAGE pHYR325L编码325位上的Arg碱基密码子CCC突检测目的蛋白纯度,收集合并酶活峰,用冻干机冻于变为Leu的密码子CTC。浓缩,用蒸馏水溶解冻干的酶蛋白,在磷酸缓冲液2.2突变基因的表达和突变酶的纯化(50mmol/L,pH6.6)中透析,再用 Superdex G75分子含有突变基因的质粒分别转化到大肠杆菌筛柱层析纯化,洗脱液为磷酸缓冲液(50mm/L,AS1.1739中,经乳糖诱导,发酵罐大量培养,目的基pH6.6)。以上步骤均在常温下进行。因得到大量表达,将表达产物分别经加热分离、硫酸123酶活测定:0.1mL4mmo/ L ONPG,0.1mL铵分级沉淀、DEAE和 Superdex G75分子筛层析纯pH58磷酸缓冲液,0.7mLH2O,混匀后于85℃水浴化,得到电泳纯,纯度均达90%以上(图1);除保温5min,加入0.1mL酶液,反应10min,加人4 Angle164Q和 TnglyE338A外,其他突变酶在在85℃mLNa2CO3溶液终止反应,420m测定吸光值。此时的比活均与野生酶相当(表2)条件下每分钟产生1pmo对硝基苯酚的酶量为一2.3突变酶与野生酶结构的比较个酶活单位。野生和突变纯酶由CD光谱仪直接扫描得到观1.24蛋白含量测定:以牛血清白蛋白为标准,察值(mdeg),把观察值换算成平均残基椭圆度(meanLowy法测定residue ellipticity)。结果显示突变酶与野生酶结构1.2.5CD光谱分析和同源建模:一定浓度的各种相同加图?因为「n半递闵曲线拥挤,这里只显示酶蛋白分别加入测量杯中,把测量杯放入光谱仪两个中国煤化工图)。说明单个氨( JASCO J-715)中,用200~250m的光进行扫描。用基酸CNMHG的折叠。同源建模蛋白质分子量和氨基酸残基数目计算残基椭圆度。结果也显示置换位点的结构域与野生酶相同(见图同源建模在htp:/w, expasy,org/ swissmod/3),这与CD光谱结果一致。杨雪鹏等:非解朊栖热菌HGI02耐热β-糖苷酶的结构与功能研究表2突变纯酶的比活Table 2 The specific activity of purified TnglysPurified proteinsE338AP316GP356AP344FP316G/P56Aecific activity/(u/mg) 17.60.9820.0M IkD5000WT T66一Tngly P316G/p356A图1 SDS-PAGE分析野生酶Tngy和突变酶的纯化20000Fig. I SDS-PAGE pattern of purifed Tnglys211: Tngly E164Q: 2: Tngly P344F: 3: Tngly P316G4: Tngly P356A: 5: Tngly R325L: 6: Tngly P316G/P356A图2突变酶和野生酶CD光谱图7: wileM: standard marker proFig. 2 Circular dichroic spectra of wild-type Tngly2.4质子供体与亲核基团的确定and two mutant TnglyR325L, Tngly P316G/P356A in the突变酶 TnglyE164Q的比活是野生酶比活的The spectra were measured on a JASCO J-715 spectrolarimeter at 25C55%,且几乎不受反应液pH值的影响(见图4);突 The protein concentration of wild-type Tngly was 0.16 mg/mL, the Tng变酶 TnglyE338A检测不到水解活性,在CMP3-F-hyB32Lwas0.053 mg/ml, the Tingly P316cP356Awas009mgmlNeu5Ac和甘露糖为底物的情况下表现出转糖苷功p6.6,50mml能(见图5),用相应的糖苷酶Tngy和a2-(3,6)-和底物形成共价复合物,是双置换反应的关键步骤NANaseⅡ都没能把反应产物水解。根据以上结果突变酶 Ingle384失去水解功能而只有转糖苷功确定Gu164为水解反应时的质子供体,介导水的攻能,可作为糖苷合成酶用于寡糖的合成。击,Gu338为水解反应时的亲核基团,其侧链羧基23314图3突变点的结构和野生酶的中国煤化工CNMHGwild-type and mutants structures are superimposed. A: Arg325L; B: P344F; C: P316G: D: P356AChinese Journal of Biotechnology生物工程学报2005,vol,2l,No.1· TnglyP3l6GiP356A图4突变酶 TnglyEI64Q和野生酶的活性受反应液pH值的影响图6突变酶的最适反应温度Fig. 4 Comparison of the ph dependence on the ONPG substrateFig. 6 EHects of temperature on site-directedwild-type Tngly and TnglyE164Q at the same condition of reactionmutagenesis Tngly hydrolysis activities1254567The hydrolysis activity was determined as describedials and Methods at differentfrom50℃to95℃Tngly316G/P356A-Highlight图5突变酶 TnglyE33A的转糖苷反应TLC图谱Fig. 5 Transglycosylation activity of mutantTngly E338A at 65C, pH6. 82: sialic acid: 3: CMP-3-F-Neu5Ac: 4: mannose and CMP-3.-Neu5Ac reacted with mutant Tngly E338A: 5: mannose and CMP-3-F.图7突变酶的最适反应pHNeusAc with no enzyme: 6: production of reaction of lane reacted withEfects of pH on site-directed mutagenesiswild-type Tngly: 7: production of reaction of laned reacted with a-2-(3Tngly hydrolysis activities6)-NANasc l (reaction of condition rely on the enzyme of optimum con-The hydrolysis activity was determined as describedin Materials and Methods at different pH from 4. 5 to 9.025热稳定性相关的突变酶酶学性质分析252最适pH值:如图7所示,6个突变酶的最适21最适温度:在50-95℃的温度范围内测定pH值都是大约pH5.8,与野生酶相似。说明置换的野生酶和突变酶的最适反应温度,发现突变酶氨基酸不影响酶活性中心的环境,也表明置换的点TnglyP34!F的最适反应温度与野生酶都为90℃,其在蛋白的外周不影响酶蛋白质的活性。它突变酶最适反应温度都有所下降(如图6),2.5.3热稳定性:突变酶和野生酶在85℃温浴不Ingly316G和 TnglyP56A最适反应温度大约为同的时间,然后测剩余酶活,当剩余酶活是原来的87℃,Tngy-P316G/P356A和 Ingly325L最适反应温50%时,各种酶经历的时间(tm)分别是野生酶Tngy度大约为85℃。结果表明位于第六 a-helix的N端63min突变酶TnyP344F57min, Ingly316G43min,第一位的P316、位于第七 a-helix的N端第一位的TngyP356和位于第六a- helix的R325置换都降低了酶蛋P35中国煤化工min和 Ingly316c/在不同的温度温白的刚性结构,但在Loop区的P34对酶的结构刚浴CNMH出相似的结果,Tm性影响较小。值是酶剩余50%活力时所对应的温度如图9,结果杨雪鹏等:非解朊栖热菌HGl02耐热β糖苷酶的结构与功能研究89力学常数,底物浓度由2mmo/L到40mmol/L。结果iyP0356Aa显示动力学常数基本相同(见表3),表明突变酶置Pro316Gl换的氨基酸不影响酶的亲和性。一Arg325Leu- Pro344Phe表3野生酶及其突变酶热稳定性和反应动力学常数Table 3 Thermodynamic and kinetic Parameters for(ken/K)▲0.010020030040050060.070.0wild-type63±0.594.0±0.5t/min图8野生酶和突变酶的热稳定性分析Pro356Ala45±0.591.5±0.5-2.5and its mutants at 85C Values whose erreArg325Leu33±0.589.0±0.5-5.0±0.578,6within 5% were averaged15±0.586.5±0.5-7.5±0.51,478.2Pro356Alaa: Determined by kinetics of irreversible heat inactivity at 85Tc: Obtained by catalytic reaction of enzyme at optimum temperature3讨论s0→ wild-type糖苷酶家族1β糖苷酶采用保留型双置换催化/ Pro356A机制,在酶的活性部位存在两个重要的羧酸部分,I Pro316Gly个质子化,称为质子供体,另一个发生离子化,称为10 -. Arg325Leu亲核基团,所切糖苷键的氧原子被质子化的羧基攻F Pro344Phe75080.0850击,糖苷键断裂,形成的碳正离子与离子化的另一个10950Incubation temperature/C羧基以离子键或共价键的形式形成中间产物,此中图9野生酶和突变酶的热稳定性分析间产物不稳定,当亲核试剂进行亲核攻击时,此中间Fig.9 Effect of temperature on the stability of产物不存在,取而代之的是一个新的糖苷键,当亲核试剂是水时,发生水解反应,当亲核试剂是醇或某个Each purified enzyme was treated at different temperatures for 15 minutes糖的羟基时,则发生转糖苷反应62。质子供体在The remaining activities were expressed as percentages of the original activities. Values whosewithin 5% were averaged双置换当中起到酸/碱催化的双功能,在第一步提供质子,在第二步介导水的攻击,亲核基团是形成显示野生酶和突变酶 TnglyP344F、 Tngly-F316、酶与底物中间物的关键氨基酸残基TnglyP356A、 TnglyR325L、 T'nglyP3l6G/P356A的T。值Agrobacterium faecalis e-葡萄糖苷酶的质子供体分别是949391、91、89和86℃。以上结果表明位和 Sulfolobus solfataricus糖苷酶两个活性位点的谷于第六α-helⅸx的N端第一位的P316和位于第七氨酸残基都已确定3·。目前,在糖苷酶家族1中α- helix的N端第一位的P356刚性结构及位于第六除了葡萄糖硫苷酶外,质子供体和亲核基团两个活a-helix的R325所形成的离子键对酶蛋白的热稳定性位点都是谷氨酸,分别在第四 B-sheet和第七p性有一定的贡献,而在Lop区的P344对蛋白质的het上。这两个活性位点氨基酸残基在家族1中很稳定性影响较小。保守,靠近蛋白质N端是T(F/LM)NE(P/L/I),靠近24突变酶米氏常数(K)和转化数(k)的测C端是(n中国煤化工当中,El64所在定:野生酶及突变酶可以水解 ONPGIe、 ONPGalTLNEPCNMHG域,分别位于第ONP-GFuc、 PNPGIc、 PNPGal、 PNPFuc和 PNPMan等多四shet相第七 B-sheet上,叮捱凋El64和E38可种底物,这里只选择ONP作为底物来测定酶的动能为糖苷酶Tngy的两个活性位点,在水解反应当Chinese Journal of Biotechnology生物工程学报2005,vl21,No.1中前者作为质子供体,后者作为亲核基团。Glu164P344的B值分别为2527、28.51和29.97。B值和和Gu38分别置换为Ghn和Aa的突变试验结论与原子的稳定性有关,B值越小原子群的稳定性越大。上述推测一致。β-糖苷酶 Tingly热稳定性可能不只是含有脯氨糖苷酶水解反应时糖苷键的水解有立体选择酸的结果,在蛋白质一级序列当中含有96%的精性,但合成糖苷键时,合成的键型是多样的,可能是氨酸,在3D结构当中发现精氨酸大部分位于蛋白B键或a键,也可能是1-3、14或16等"。耐热突质外周,在a- helix之间形成离子键网络。这样的离变酶 TnglyE338A在65℃温度下可催化底物CMP3-子键在其他耐热蛋白质中都被证明为重要的稳定因F-Neu5AC和甘露糖的合成,合成产物不能用相应的子0,别。较早Pent等人比较了同一种酶不同糖苷酶Tngy和a-2-(3,6)- NANase水解,表明产物来源的耐热酶和常温酶的结构发现,耐热酶的分子的糖苷键键型可能发生了改变。这样产生的混合键表面离子键比常温酶的多。在以后的耐热酶研型连接的寡糖( mixed-linkage)有可能作为寡糖类似究中也发现类似的情况,比如,苹果酸脱氢酶、甘物用于糖苷水解酶的抑制剂1油醛-3-磷酸-脱氢酶和DNA聚合酶琍等。我们蛋白质的耐热因素包括离子键作用、氢键作把6a-heix的Amg325和5 a-helix的A甲p235之间形用、疏水作用、金属键、二硫键、包装效应、Poie理成的离子键打断,突变酶R2SL的热稳定性参数T论、a螺旋的稳定作用和氨基酸组成等都经过深入值下降大约5℃,表明蛋白质外周的离子键对野生广泛的分析3,从大量的研究中发现,一个蛋白酶Tngy热稳定性也有贡献。质的耐热机制可能是多种因素的结果。各个突变酶与野生酶纯化方法相同,从SDSpone与其他的a氨基酸不同,由于其N原子PAGE电泳结果分析各个突变酶与野生酶纯度相位于吡咯环上,使得前一个氨基酸与它形成肽键时当,然而突变酶 TnglyP316G、 TnglyP356A、 Ingly316G(C-N)不能自由旋转,另外吡咯环还具有疏水作P56A和 TnglyR32L的比活与野生酶相比有所上用。Poue分子的这些特点,在形成肽链时导致它升,可能的原因是在85℃时突变酶结构与野生酶相比其它氨基酸的构型熵小22.2,从而降低蛋白质比“刚性”下降“柔韧性”增加,有利于突变酶催化反的折叠熵。蛋白质的折叠熵降低可以提高其稳定应。突变酶 TnglyP316G、 Ingly356A、 Ingly3l6G性33。较早,从统计的结果表明蛋白质中脯氨酸含量增多,可以明显提高蛋白质的热稳定性),但P356A、 Ingly344F和 Ingly325L动力学常数与野生酶基本相同,说明被置换的氨基酸残基与酶的活性脯氨酸在蛋白质二级结构中的位置不同对稳定性的贡献不一样,动力学模拟试验证明脯氨酸位于a无关,置换上来的氨基酸残基也不影响酶活性中心heix的N端第一位最有利于蛋白质的稳定性结构的正确折叠。统计结果也表明脯氨酸偏向位于α- helix的N端第 REFERENCES(参考文献)位和Bum的第二位,这可能有利于蛋白质的[1] Ichikawa Y, Look GC, Wong CH. 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