糖基轉(zhuǎn)移酶與糖苷酶

1、第二節(jié)糖基轉(zhuǎn)移酶及其應(yīng)用糖基轉(zhuǎn)移酶及其應(yīng)用主要內(nèi)容主要內(nèi)容l糖轉(zhuǎn)移酶簡介 l糖基轉(zhuǎn)移酶在合成中的應(yīng)用 Glycosyltransferases 應(yīng)用activated sugar phosphates 作為糖供體，合成 glycosidic linkage ，糖受體通常為 nucleophilic group, usually an alcohol. 生成的糖苷可以為 O-, N-, S-, or C-glycoside 糖轉(zhuǎn)移酶簡介糖轉(zhuǎn)移酶簡介功能：催化糖苷鍵的合成（功能：催化糖苷鍵的合成（ O-, N-, S-, or C-glycoside ）供體：活化的糖磷酸供體：活化的糖磷酸受體：親

2、核基團（蛋白、脂、核酸、糖、小分子），通常為受體：親核基團（蛋白、脂、核酸、糖、小分子），通常為-OHNon-Leloir donors：Leloir donors：糖供體底物糖供體底物糖基轉(zhuǎn)移酶糖基轉(zhuǎn)移酶根據(jù)根據(jù)糖供體糖供體中是否含有中是否含有核苷酸核苷酸分為分為兩類兩類。Leloir:阿根廷生物化學家，研究核苷酸代謝，1970，諾貝爾化學獎l 分為94 家族 （distinct sequence-based families） (CAZy server, http:/rs-mrs.fr/CAZY)l 人類擁有約 270多種糖基轉(zhuǎn)移酶序列，屬于 33個家族。l Alpha-1,4-葡萄糖轉(zhuǎn)移

3、酶；beta-1,4-半乳糖轉(zhuǎn)移酶；2,3-唾液酸轉(zhuǎn)移酶糖基轉(zhuǎn)移酶分類糖基轉(zhuǎn)移酶分類根據(jù)氨基酸序列相似性進行分類：根據(jù)氨基酸序列相似性進行分類：根據(jù)蛋白結(jié)構(gòu)相似性進行分類：根據(jù)蛋白結(jié)構(gòu)相似性進行分類：根據(jù)糖供體和糖苷鍵連接方式進行分類：根據(jù)糖供體和糖苷鍵連接方式進行分類：l GT-A，GT-B，其他類型Rossmann-type domains (for nucleotide binding)NDP-binding domain generally contains a conserved DXD amino acid motifGT-A fold: SpsA from Bacillus su

4、btilusGT-B fold: beta-glucosyltransferase from bacteriophage T4Leloir GTsTransglycosylase from Staphylococcus aureusOligosaccharyltransferase STT3 from Pyrococcus furiosiusNon-Leloir GTs催化機理催化機理 Glycosyltransferases catalyze the transfer of glycosyl groups to a nucleophilic acceptor with either rete

5、ntion or inversion of configuration at the anomeric centre. This allows the classification of glycosyltransferases as either retaining or inverting enzymes. 鎓 催化機理催化機理Inverting: SN2 nucleophilic attack at the C1 atomRetaining: double displacement mechanism 糖基轉(zhuǎn)移酶輔因子糖基轉(zhuǎn)移酶輔因子l Many, but not all, glycos

6、yltransferases utilize divalent metal ion cofactors such as Mn2+ and Mg2+. mainly in glycosyltransferases that are diphosphonucleoside-dependent. l metal ion is coordinated to an oxygen of each of the two phosphate groups, as well as to side-chain carboxylates derived from the protein. 糖基轉(zhuǎn)移酶抑制劑糖基轉(zhuǎn)移酶

7、抑制劑 直接抑制糖基轉(zhuǎn)移酶活性直接抑制糖基轉(zhuǎn)移酶活性底物類似物；過渡態(tài)類似物alpha-2,6-唾液酸轉(zhuǎn)移酶抑制劑Beta-1,4-半乳糖轉(zhuǎn)移酶抑制劑 阻斷糖供體的合成阻斷糖供體的合成N-Glycan 合成過程中，首先要合成：dolichol-pp-N-acetylglucosamineUDP-GlcANc + dolichol-p - dolichol-pp-GlcNAcN-acetylglucosamine phosphorotransferase糖基轉(zhuǎn)移酶在合成中的應(yīng)用糖基轉(zhuǎn)移酶在合成中的應(yīng)用 寡糖的酶法合成寡糖的酶法合成 具有生物活性含糖天然產(chǎn)物的酶法合成具有生物活性含糖天然產(chǎn)物的酶法

8、合成 生物制藥生物制藥-糖蛋白藥物生產(chǎn)策略糖蛋白藥物生產(chǎn)策略 生物制藥生物制藥-糖疫苗生產(chǎn)策略糖疫苗生產(chǎn)策略O(shè)AcOAcOOAcOAcOOAcOAcOOAcOAcOHOOAcOAcOOAcOAcOOAcN3OOAcOOAcOAcOOAcOAcOOAcN3OBnOBnOBnOOBnOOHOOHOHOOHOHOOHNHAcOHOHOHOOH2679N33 steps6 stepsglycosylation4 stepsChemical Synthesis of a-Gal規(guī)模小，過程復(fù)雜，立體選擇性難規(guī)模小，過程復(fù)雜，立體選擇性難OOHHOHOHOO P O PONNHOOOOHHOHOHOOOH

9、HONHAcOOROOHHOHOHOOOHHOOOHOOHHONHAcOOROOOO-O-HO P O PONNHOOOOOO-O-OHHOOHHOGal1,4GlcNAc-R, acceptor-Gal epitopeUDPUDP-Gal Donor1,3-galactosyltransferaseMn2+Reaction Catalyzed by 1,3-Galactosyltransferase碳水化合物的合成碳水化合物的合成 Organic Synthesis Biosynthetic process Derivative synthesis Versatile Not very fl

10、exible Process Expensive and multistep Economic and short Environment problem Chemical waste Environment-friendly Carbohydrate polymer synthesis Not applicable Practical approach 體內(nèi)糖供體合成途徑體內(nèi)糖供體合成途徑 Glc, GlcNAc & ManSugar-6-pSugar-1-pSugar-NDPkinaseMutasepyrophorylase Gal, GalNAc & FucSugar-1

11、-pSugar-NDPkinasepyrophorylase Neu5AcNeu5AcCMP-Neu5AcpyrophorylaseCTP 糖供體相互轉(zhuǎn)化: GalE: UDP-Gal UDP-Glu GalNAcE: UDP-GalNAc UDP-GlcNAc UGD(UDP-Glc dehydrogenase)： UDP-Glc UDP-GlcA UDP-GlcA decarboxylase: UDP-GlcA UDP-Xyl糖基轉(zhuǎn)移酶介導(dǎo)的寡糖合成糖基轉(zhuǎn)移酶介導(dǎo)的寡糖合成 PhosphorylasesGlucosyltransferasesFructosyltransferasesCyc

12、lodextrin glucanotransferasesMany微生物中的糖供體微生物中的糖供體天然糖苷化合物中的糖一般為 C2, C3, C4 and/or C6 脫氧糖，而且大部分為6-deoxyhexoses family （Rhamnose）原核生物糖轉(zhuǎn)移酶具有糖供體糖供體的廣泛性 6-deoxyhexoses 一般通過 TDP-sugars進行底物活化（TDP-Rhamnose）優(yōu)點優(yōu)點: * 區(qū)域、立體選擇性* 大量生產(chǎn)不足不足: * 糖基轉(zhuǎn)移酶在大量表達方面存在困難 糖供體比較昂貴糖供體比較昂貴. 副產(chǎn)物抑制消除副產(chǎn)物抑制（堿性磷酸酶）消除副產(chǎn)物抑制（堿性磷酸酶）原位產(chǎn)生糖供體

13、原位產(chǎn)生糖供體 .針對以上不足解決策略針對以上不足解決策略糖基轉(zhuǎn)移酶在工業(yè)應(yīng)用中優(yōu)勢與弱點糖基轉(zhuǎn)移酶在工業(yè)應(yīng)用中優(yōu)勢與弱點可作為LgtC底物 堿性磷酸酶酶法再生糖供體策略酶法再生糖供體策略 UDP-sugar (A) and CMP-sugar (B)糖供體的合成糖供體的合成糖供體的合成糖供體的合成糖供體的合成糖供體的合成糖基轉(zhuǎn)移酶在糖基轉(zhuǎn)移酶在寡糖合成中的應(yīng)用寡糖合成中的應(yīng)用（一）（一）具有生物活性寡糖的酶法合成具有生物活性寡糖的酶法合成(for details visit: )OHOOHHOHOOHOOHOHOOOOHHONHACOHOOHOHOOOO

14、HHOHOResearch on Carbohydrates in Human Immunity in Wangs LaboratoryOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBlood type B antigenType B bloodType A bloodAnti-B antibody Barrier in blood transfusion Barrier in organ transplantation Cause of hyperacute rejection in animal to human xenotransplantation

15、 Human natural immunity against pathogens, tumors, etc.Non-primate mammals(pig, mouse, dog etc.)New world monkeyHumanApeOld world monkeyAnti-Gal antibody(the most abundant natural Ab)1-2% total IgG, 3-8% total IgM -Gal epitopeabundant, 1 million/cellHuman - HumanHuman - AnimalOHOOHOHOHOOHHOHOOHOOHOH

16、OOOOHHONHACOHOOHOHOOOOHHOHOThe specificity of glycosyltransferases determine the sugar sequenceOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBlood type B antigenOHOOHOH -Gal antigenOOHOHHOOOOOHHONHACOHOOHOHOOOOHHOHOOHOOHOHOOHOHHOHOOOOHHONHACOHOOHOHOOOOHHOHOhuman 1,3GalTpig 1,3GalTAnti-Gal productionmill

17、ion years ago 5-15 20-30 30-40 60-70 70-80120-130mammals -gal epitopesynthesishumansapesmonkeysOld WorldNew WorldmonkeysprosimiansmarsupialsnonprimateplacentalmammalsInactivation of 1,3 GTBiomedical use of -Galu soluble -Gal antagonist to anti-Galu -Gal conjugates in immunotherapy against bacteria,

18、virus, and cancer cells.人工合成的必要性人工合成的必要性O(shè)OHHOHOHOO P O PONNHOOOOHHOHOHOOOHHOOHOOROOHHOHOHOOOHHOOOHOOHHOOHOOROOOO-O-HO P O PONNHOOOOOO-O-OHHOOHHOReaction Catalyzed by 1,3-GalactosyltransferaseGal1,4Glc-R, acceptor-Gal epitopeUDPUDP-Gal Donor1,3-galactosyltransferaseMn2+1,3GalT 催化合成 -Gal Epitopes 及其衍生

19、物HOOOHHOHOOUDPHOOOHHOHOOUDPOHOOHHOOOOHHOR1R2HOOHOOHHOHOOHOOHOHOOOOHHOR1R2UDP-Gal4-epimeraseUDP 1,3GalTR1R2OHOHOHOHNHAcN3SPhOAllylabcdNHAcOHeNH2OHfUDP-Gal100mg/$416 UDP-Glc5g/$529.5EC Fang J, Chen X, Wang PG, et al.: J. Org. Chem. 1999, 64, 4089-4094. OOHOHOHOOOHOHHON3OHOOOHNHAcHOOOHOHOHOOOHOH

20、HON3OOOOHNHAcHOOOH OHOHOOOHOHOHHOOOHOHOHOOOHOHHON3OOOOHNHAcHOOOH OHOHHOHOOOHHOHOOUDPHOOOHHOHOOPO3=OPO3H-COOHOCOOH2PiUTPUDP-Gal4-epimeraseUDP1,4 galactosyltransferase1,3 galactosyltransferasePPiUDP-GlcUDP-GlcPyrophospho-rylasePyruvateKinaseInorganicPyrophos-phatase通過原位再生合成 a-Gal 五糖Fang J, Li J, Chen

21、X, Wang PG, et al: J. Am. Chem. Soc. 1998, 120, 6635-6638.其他生物活性寡糖的合成（應(yīng)用）其他生物活性寡糖的合成（應(yīng)用）In Human milkOOHOHOOHOOHOHOOHOHHOOOHOOOHOHOHOOH1,4GalTlgtC gene(a) globotriose Gb3OHOHOAcHNOHOOHOHOOHOOHOHOOHOHOHOOHOOH1,3GalNAcTlgtD gene(b) globotetrose Gb4OHOOHOOHOOHOHOOHOHHOOHOAcHNOH(c) LNT-21,3GlcNAcTlgtA g

22、eneOHOOHOOHOOHOHOOHOHOOHOAcHNOH(d) LNnT: Lacto-N-neotetraose1,4GalTlgtB geneHOOHOHOOHOHOOHOOHOOHOHOOHOHOHOAcHNOH2,3transialidase(e) LSTDOHOAcHNHOOHHOHO2COH(Without the B subunits, the A subunit has no way of attaching to or entering the cell, and thus no way to exert its toxic effect.)Stx is an AB5

23、subunit toxin. The pentamer of (small) B subunits binding to its receptor glycosphingolipid(GSL), globotriaosyl ceramide(Gb3) in glomerular endothelial cell membranes, initiates A subunit-mediated cell death leading to HUS（hemolytic uremic syndrome ）, but induction of inflammatory pathways is also k

24、ey. Gb3 is heterogenous in its lipid structure and membrane organization, such that different Gb3 formats are differentially recognized by Stx family members, particularly Stx2, which is more frequently associated with clinical disease. 血型相關(guān)抗原酶法合成血型相關(guān)抗原酶法合成Fluorescently labeled sLex conj sLex在轉(zhuǎn)移性結(jié)腸癌

25、高表達轉(zhuǎn)移性結(jié)腸癌高表達二價腫瘤抗二價腫瘤抗原酶法合成原酶法合成（用于檢測腫瘤位置）（用于檢測腫瘤位置）糖基轉(zhuǎn)移酶應(yīng)用乳腺癌、前列腺癌上高表達Globo-HP-凝集素凝集素 配體配體防治黑色素瘤防治黑色素瘤l 寡糖的合成常常需要多種糖基轉(zhuǎn)移酶寡糖的合成常常需要多種糖基轉(zhuǎn)移酶l 為了增加產(chǎn)量還有用其他一些酶為了增加產(chǎn)量還有用其他一些酶l 分步合成費時費力分步合成費時費力？一鍋多酶法一鍋多酶法- Super beads - Super bugJ. Am. Chem. Soc. 1995, 117, 5869-5870一鍋多酶法合成透明質(zhì)酸 （hyaluronic acid）E1, hyaluron

26、ic acid synthase; E2, UDP-Glc dehydrogenase; E3, UDP-Glc pyrophosphorylase; E4, UDP-GlcNAc pyrophosphorylase; E5, pyruvate kinase; E6, lactate dehydrogenase; E7, inorganic pyrophosphatase OHOHOHOOHOOHOHOOROHOHOOHOOOOHOOROHOHOHOAcHNHOOHHOHO2CCO2HOHOAcHNHOOHHOOCMPPPiPEPE3HOOHOOHOHOHE4E5HOOHOOHOHOPHOOH

27、OOHOGDPOHGDPPyruvateGTPHOOHOOHOPOHOGDPOOHHOCH3OHNADPH/NADHNADP/NADGlcAGlcE1E2E6E7E8OOHHOCH3OHOHOHOHOOOOHOOROHOHOOHHOCH3OHCMPSialyl Lewis X antigenLewis X antigensialyl Lewis X 酶法大量合成酶法大量合成E1: 1,3-fucosyltransferase；E2: pyruvate kinase；E3: GDP-mannose pyrophosphorylaseE4: GDP-4-keto-5-deoxymannose 3,

28、5-epimerase/GDP-4-keto-6-galactose reductase； E5: glucose dehydrogenase； E6: hexokinase； E7: phosphomannomutase； E8: a2,3-sialyltransferase激酶激酶歧化酶歧化酶SuperbeadGalUGalTGalKPyKFRecombinant E. coli stratins overexpressingGalK, GalT, GalU or PykF1) Fermentation2) Lysis3) Add to Ni2+ resinChen X, Fang J,

29、Wang PG, et al.: J. Am. Chem. Soc. 2001, 123, 2081-2082.Beads with:GalKGalTGalUPykFPeristaltic pumpfor circulationReservoir with: 1,3GalTLacOBn 9.6 mMATP 0.96 mMPEP 19.2 mMUDP 0.96 mMGlc-1-P 0.96 mMGal 12 mM MgCl2 10 mMMnCl2 10 mMKCl 100 mM HEPES 100 mM pH 7.5Liu Z, Zhang J, Chen X, Wang PG: ChemBio

30、Chem 2002, 3, 348-355.Production of UDP-Gal with Superbeads. Superbead 用于寡糖合成OHOOHOHOOOOHHONHAcOHOHOOHHOHOOHOOHOHOOHOOHHOHOOMeOOOHHOHOOHOOHOHOOHOOHHOOBnOOOHHOHOOHOOHHOHOOOOHHONHAcOHOOOHHOHOOHOOHHOHOOBnOOOHHOHOOHOOHOHOOOOHHONHAcOOHOOHHOHOOHOOHHOOOOHHOOHN3OHOOHOHOOHOOHHOHOOBnOOOHHOHO1,3GalT85%(a72%)83

31、%69%92%95%1,4GalT1,4GalT86%76%1,3GalT + 1,4GalTa Gram scale synthesis, others are 100 mg scales從多菌種發(fā)酵到從多菌種發(fā)酵到Super bugUMPUDPUTPGlobotioseUDPUDP-GalOrotic AcidUTPPPi2PiGlc-1-PUDP-GlcUDP-GalGal-1-PGalactose (100 g/L)GalKGalTGalUGlucoseFructoseC. ammoniagenes DN510E. coli NM522/pNT25/pNT32Globotriose(G

32、al1,4Lac)188 g/L, 36 hr.LgtCE. coli NM522/pGT5PpaLactose (100 g/L)Kyowa Hakkos technology for large-scale production of UDP-Gal and globotriose utilizing metabolically engineered bacterial cells OOHOHOOHOOHOHOOHOHOHOHOHOOHglobotriose Gb3Ppa: pyrophosphataseGalU: glucose-1-phosphate uridylyltransfera

33、seGalT: galactose-1-phosphate uridylyltransferaseGalK: galactokinaseLgtC: 1,4-galactosyltransferaseCMP-NeuAc regeneration system by bacterial coupling Using this approach, sugar nucleotides including UDP-Gal,CMP-Neu5Ac, UDP-GlcNAc and GDP-Fuc have been successfully produced on a large scale.Superbug

34、 technologies for the synthesis of -GalBacteriaNutrientsStartingMaterialsBio-synthetic pathway engineeringa-Gal分析合成途徑分析合成途徑ATPADPGalK : galactokinaseGalUT: galactose-1-phosphate uridylytransferaseGalU : glucose-1-phosphate uridylyltransferasePykF: pyruvate kinase1,3GalT: 1,3-galactosyltransferase1,3

35、GalT Gal1,3LacL actoseUDP-Gal UDPPEPPyruvateGal-1-PGlc-1-PUDP-GlcUTPPykFGalUGalKGalUTGalactosePPi克隆表達驗證相關(guān)酶克隆表達驗證相關(guān)酶 Clone and express individual enzymes in the biosynthetic pathway of -Gal （克隆相關(guān)酶） Construction an artificial biosynthetic gene cluster and transfer into E. coli host cell.（構(gòu)建基因簇并轉(zhuǎn)化） Lar

36、ge scale production of -Gal oligosaccharides using fermented and permeated cells. （發(fā)酵生產(chǎn)） EnzymeAmount (U/L) 1,3GalTGalKGalTGalUPykF 6025100100501 U = 1 mol / min, 37oCpLDR20-KTUFgalK + galT1,3GalTgalUpykFT7 terminatorl l PRpLDR20-KTUFAmprEcoR VSac IISal IXba ICla I 9 kbl cI - repressorpMB1rbcrbcrbcr

37、bc構(gòu)建多基因表達質(zhì)粒構(gòu)建多基因表達質(zhì)粒檢驗多基因表達效果檢驗多基因表達效果66,20031,00021,50045,000LysateMIBMPure14,50097,400PykFGalKGalTGalU1,3GalTSuperbug 整個生產(chǎn)過程 Whole Cell ReactionAnalyzed by TLC, HPLCRt,20-36h Stop by inserting in boiling waterCentrifugationGel filtration P21H NMR, 13C NMR Analysis Culture NM522/pLDR20-KTUF Centrif

38、ugationOverexpressionResuspended inTris-HCl BufferE . coli N M 522pL D R 20-K T U F -G al7.2 g Gal1.1 g ATP8.4 g Lac3.6 g GlcTotal volume: 1 L 0.3 g UDP-Glc7.2 gChen X, Liu Z, Zhang W, Fang J, Andreana P, Wang PG: ChemBioChem. 2002, 3, 47-53.大規(guī)模發(fā)酵產(chǎn)量大規(guī)模發(fā)酵產(chǎn)量Superbug-11,3GalTGal1,3Gal1,4GlcGal1,4GlcUDP

39、-GalUDPPEPPyruvateGal-1-PGlc-1-PUDP-GlcUTPPykFGalUGalKGalTGalactoseLactoseGlycolyticpathwayGlucoseATPADPGlucosePyruvatePykFPEPGlycolytic pathwayPPiNM522添加三種三種原料：glucose, galactose, lactosePnPn-11,3GalTGal1,3Gal1,4GlcGal1,4GlcUDP-GalUDPGal-1-PGlc-1-PUDP-GlcUTPGalUPpKGalTGalactoseLactoseGalactoseNM522

40、PnPnPn-1PpKPnPPiPpK: polyphosphate kinaseSuperbug-2添加三種三種原料：多聚磷酸, galactose, lactoseHOOOHHOHOOUDPHOOOHHOHOOUDPOHOOHHOOOOHHOHOHOOHOOHHOHOOHOOHOHOOOOHHOHOOROR1,3GalTUDPSucroseFructose(energy source)SusAGalESucroseLacORE. coliChen X, Zhang J, Kowal P, Andreana P, Wang PG: J. Am. Chem. Soc. 2001, 123, 8

41、866-8867.SusA: sucrose synthaseGalE: UDP-Gal 4-epimerase添加兩種兩種原料：sucrose, lactoseSuperbug-3每次進步只有一每次進步只有一點點，積累多了點點，積累多了才會實現(xiàn)自己的才會實現(xiàn)自己的目標。目標。二二具有生物活性的含糖天然產(chǎn)物及其酶法合成具有生物活性的含糖天然產(chǎn)物及其酶法合成含糖天然產(chǎn)物含糖天然產(chǎn)物含糖天然產(chǎn)物含糖天然產(chǎn)物諾加霉素阿克拉霉素(安樂霉素)；蒽環(huán)類抗癌藥，它能嵌入癌細胞的DNA上，抑制核酸的合成，特別是RNA的合成，為周期非特異性藥物，在G1晚期和S晚期阻斷細胞周期。daunorubicin n. 微

42、道諾霉素；紅比霉素；正定霉素；柔毛霉素（用作抗腫瘤藥）含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成The glycosyltransferase UrdGT2 establishes both C and O glycosidic bonds 天然抗生素竹桃霉素天然抗生素竹桃霉素(landomycin)抗菌性與紅霉素類似抗菌性與紅霉素類似含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成含糖天然產(chǎn)物酶法合成OOHOHOObaicaleinOHOHOHOHOOOOHOOOHOHluteolin 7-glucosideOHOHOHOOHOOOHOHHO

43、OHOHOOHOOHOOHOHOHOOHquercetagetin-7-arabinosyl-galactosideOOOOHHOOHOHOMeOHOHHOquercetrinOOOHOHOOHOHOMeHOOOHOHOHOHOOHrutin 抗菌黃酮苷化合物抗菌黃酮苷化合物 OOHHOOOHOHOquercetin-3-galactosideOHOHOHOOHOOHHOOOHOHOHOmyricetin-3-O-galactosideOHOHOHOOHOOHHOOOHOHOHOmyricetin-3-O-glucosideOOHHOOHOH抗腫瘤黃酮苷化合物抗腫瘤黃酮苷化合物 OOHHOOO

44、HOkaempferol 3-o-glucosideOOHHOOHOHOOHOOOHOOHOH3CHOOHHOHOOHOapigenin 7-O-rutinosideOOHHOOOHOHOHOmyricetin-3-galactosideOHOHOHOOHOOHHOOOHOHOHOOHOHOHOmyricetin-3-arabinosideOOHHOOOHOHOquercetin 3-galactosideOHOHOHOOHOOHHOOOHOHOquercetin 3-xylosideOOHHOOHOHHOO+OHOHOcyanidin 3-galactosideOHOHOHOOH降血脂黃酮苷

45、化合物降血脂黃酮苷化合物 研究表明，對于某些黃酮藥物，糖的存在與否會對藥糖的存在與否會對藥效影響很大效影響很大。例如： diosmin，gossipyn和rutin對于中樞神經(jīng)系統(tǒng)有明顯的鎮(zhèn)靜作用，但相應(yīng)的苷元沒有類似作用。桔皮素（tangeratin）是一種非常有效的抗腫瘤細胞增生的藥物，體外研究表明包括桔皮素在內(nèi)的三種黃酮類藥物它們的糖苷型比苷元型抗腫瘤活性更高。在對乳腺腺癌細胞抑制研究中，發(fā)現(xiàn)柚皮苷、蘆丁、芹菜素、山奈酚、白楊素的抑制活性逐漸減弱，含有糖配體的糖苷類物質(zhì)活性比其他的要高些。 European journal of pharmacology, 2006，539(3): 16

46、8-176 A comparison of the structures among these compounds indicates the following elements are essential to the potential acceptors of UGT78D1: 1) the 3-hydroxyl group, 2) the double bond in the ring C, 3) the 4-hydroxyl group .Guangxiang Ren, Lianwen Zhang, Glycoconj J (2012) 29:425432酶（酶（UGT78D1）

47、的底物選擇性）的底物選擇性l 阿糖胞苷，急性白血病，糖基部分在抑制核酸代謝物與酶的結(jié)合抑制核酸代謝物與酶的結(jié)合反應(yīng)中起重要作用，從而增強了其抗腫瘤效應(yīng)。l 烯二炔類抗腫瘤抗生素通過糖基部分與通過糖基部分與DNA 結(jié)合結(jié)合，使藥物分子嵌入DNA 雙螺旋的小溝中(特別是TCCT位點)。 l 蒽環(huán)類抗生素，環(huán)A 和氨基糖部分在決定藥物與氨基糖部分在決定藥物與DNA的選擇性結(jié)合的選擇性結(jié)合中起關(guān)鍵作用中起關(guān)鍵作用。該類藥物與DNA作用形成復(fù)合物的穩(wěn)定性主要決定于藥物的糖環(huán)與DNA之間的作用。以以DNA為靶點的抗腫瘤藥物為靶點的抗腫瘤藥物糖配基具有協(xié)助藥物生物轉(zhuǎn)運和特異性識別并結(jié)合協(xié)助藥物生物轉(zhuǎn)運和特異

48、性識別并結(jié)合DNA 的作用的作用。l 含糖天然產(chǎn)物或藥物大量存在l 糖分子在藥物分子執(zhí)行其功能時有時具有重要作用l 人工添加糖分子存在一定困難選擇合適的藥物分子或改造某種功能已知的糖苷藥物，設(shè)計合成途徑選擇合適的藥物分子或改造某種功能已知的糖苷藥物，設(shè)計合成途徑小結(jié)如下：小結(jié)如下：三：糖蛋白藥物三：糖蛋白藥物正在開發(fā)的蛋白質(zhì)正在開發(fā)的蛋白質(zhì)和抗體藥物中大約和抗體藥物中大約83%是糖蛋白。是糖蛋白。 2000-2005(%) 2005-2010(%)抗體抗體 35 25蛋白蛋白 15 12Small Molecules 8 8Revenue In Billions200020052010預(yù)計到預(yù)

49、計到 2010年，年，糖蛋白藥品市場可糖蛋白藥品市場可達達680億美元億美元 。國際生物藥品市場占有率分析國際生物藥品市場占有率分析 蛋白糖基化的作用Protein foldingProtein targeting/traffickingLigand recognition/bindingBiological activityStabilityRegulates protein half-lifeImmunogenicityglycocomponent of glycoproteins美國美國Amgen公司的公司的EPOGEN（EPO, 紅細胞生成素）紅細胞生成素）和和Aranesp（最新的紅

50、細胞生成素）：（最新的紅細胞生成素）：EPOGEN，包含有，包含有165個氨基酸，其序列與天然的內(nèi)個氨基酸，其序列與天然的內(nèi)源性紅細胞生成素相同，具有相等的生物活性。源性紅細胞生成素相同，具有相等的生物活性。Aranesp是第二代紅細胞生成蛋白，比是第二代紅細胞生成蛋白，比EPOGEN多兩多兩條條N-糖鏈，包含了更多的唾液酸，其體內(nèi)糖鏈，包含了更多的唾液酸，其體內(nèi)半衰期比半衰期比EPOGEN長長3倍多倍多，從而顯示出更強的生物活性。，從而顯示出更強的生物活性。目前糖蛋白表達的缺點和不足非人源化的糖蛋白非人源化的糖蛋白：糖蛋白的快速清除，改變藥代動力學性質(zhì)，補體激活，提高免疫原性和用藥安全問題。

51、現(xiàn)有人源化的糖蛋白現(xiàn)有人源化的糖蛋白：生產(chǎn)成本高，細胞容易污染，N-糖基化不足，糖基化程度不同（同一批次和不同批次）。Sethuraman N, Stadheim TA. Challenges in therapeutic glycoprotein production. Curr Opin Biotechnol. 2006. 17(4):341-6. l 摻入非天然氨基酸摻入非天然氨基酸l 特定化學反應(yīng)引入糖基修飾特定化學反應(yīng)引入糖基修飾l 細胞表達復(fù)雜糖修飾蛋白細胞表達復(fù)雜糖修飾蛋白 l 特定酶切（特定酶切（Endo-H）、酶連）、酶連（Endo-A/M）引入均一糖鏈）引入均一糖鏈四：糖疫

52、苗四：糖疫苗 病原微生物耐藥性逐漸變強，需病原微生物耐藥性逐漸變強，需要更安全有效的防治方法要更安全有效的防治方法 多種微生物表面成分可作為疫苗多種微生物表面成分可作為疫苗In principle, various types of cell surface epitopes, characteristic for the invading organism or related to aberrant growth of cells, can be applied to develop vaccines. 糖合成技術(shù)的出現(xiàn)為糖疫苗的研究糖合成技術(shù)的出現(xiàn)為糖疫苗的研究應(yīng)用提供了可能應(yīng)用提供了可

53、能The progress in establishing the structure of carbohydrate immuno-determinants in conjunction with improvements in carbohydrate synthesis has rendered it feasible to develop new generations of carbohydrate-based vaccines.部部分分已已知知糖糖蛋蛋白白抗抗原原糖疫苗結(jié)構(gòu)糖疫苗結(jié)構(gòu)Carrier protein:KLH (key hole Limpet hemocyanin)DT

54、 (diphtheria toxoids，CRM197)TT (tetanus Toxoids)antigen protein Glycan (起免疫作用的只是糖鏈的一部分；oligosaccharides- 有機合成或多糖降解獲得)CouplingBifunctional spacer molecules (amino, carboxyl or thiol groups)Glycoconjugate vaccines against bacterial infections 基于細菌莢膜多糖上的片段，如下列病原微生物：基于細菌莢膜多糖上的片段，如下列病原微生物： 研究內(nèi)容：糖鏈長度與疫苗功能

55、；糖鏈數(shù)目與免疫功能研究內(nèi)容：糖鏈長度與疫苗功能；糖鏈數(shù)目與免疫功能Polysaccharide type-3 related di-,tri-and tetrasaccharride -CRM197 conjugates: S. pneumoniae type 3 infectionoligosaccharide/proteinratios肺炎球菌奈瑟氏腦膜炎球菌流感嗜血桿菌傷寒沙門氏菌痢疾志賀氏菌B型鏈球菌肺炎桿菌偶聯(lián)多條糖鏈更有效偶聯(lián)多條糖鏈更有效病原微生物糖疫苗病原微生物糖疫苗 抗寄生蟲疫苗制備更復(fù)雜抗寄生蟲疫苗制備更復(fù)雜. . Malaria(Malaria(瘧疾瘧疾), ), to

56、xoplasmosis toxoplasmosis（ 弓形體病）弓形體?。?and leishmaniasand leishmanias（利（利什曼蟲）什曼蟲）. Malaria caused by Plasmodiumfalciparum. Malaria caused by Plasmodiumfalciparum（瘧原蟲）（瘧原蟲）, , 靶點：對應(yīng)前兩者為靶點：對應(yīng)前兩者為glycosylphosphatidylinositol anchors(GPIs)glycosylphosphatidylinositol anchors(GPIs)偶聯(lián)于偶聯(lián)于KLH獲得疫苗，對瘧疾有很好的防治效

57、果。獲得疫苗，對瘧疾有很好的防治效果。腫瘤糖疫苗腫瘤糖疫苗至今至今FDA只批準了一個預(yù)防肝炎病毒的疫苗只批準了一個預(yù)防肝炎病毒的疫苗糖相關(guān)腫瘤抗原糖相關(guān)腫瘤抗原Muc-1NER-2/neuCEAP53STn Globo H糖苷酶及其應(yīng)用糖苷酶及其應(yīng)用l Glycoside hydrolases (Glycosidases)l Transglycosylasesl Lyasesl phosphorylases糖鏈糖鏈 或或 糖綴合物糖綴合物糖苷鍵天然水解時，半衰期為470萬年；糖苷酶可以提高催化效率10的17次方倍。糖苷鍵切割相關(guān)酶糖苷鍵切割相關(guān)酶糖苷鍵底物及效率糖苷鍵底物及效率糖苷酶分類糖苷酶

58、分類l 按水解機理： 保持型&翻轉(zhuǎn)型l 按糖苷鍵位置： 內(nèi)切酶內(nèi)切酶，外切酶（非還原端外切酶非還原端外切酶，還原端外切酶）l 包括近13萬條糖苷酶序列，分屬130個家族 ( sequence-based families, http:/rs-mrs.fr/CAZY)l 超過30種糖苷酶的晶體結(jié)構(gòu)已經(jīng)解析l Transglycosidases (transglycosylases): 在寡糖和多糖之間進行糖基化反應(yīng) 。根據(jù)序列進行分類根據(jù)序列進行分類（EC 3.2.1.-）某些保持型糖苷酶（唾液酸轉(zhuǎn)移酶），活性中心可以包含一個酪氨酸某些保持型糖苷酶的酶促反應(yīng)中，有鄰基參與過程Myrosi

59、nases (芥子苷酶)Lyases(裂解酶)通過消去反應(yīng)的機制（eg: a-glucan lyases）Phosphorylase & Transglycosidasesl A-R+H3PO3-A-H2PO3 + RHl “磷解”糖苷鍵l 產(chǎn)物：sugar-1-P(retention or inversion)l 通常為外切酶（如glycogen phosphorylase)）l 經(jīng)過糖苷-酶過渡態(tài)，糖的羥基親和進攻合成新的糖苷鍵l “糖解”糖苷酶（環(huán)糊精合酶）TransglycosidasesPhosphorylase水解機理水解機理都要經(jīng)歷鎓離子過渡都要經(jīng)歷鎓離子過渡態(tài)；而且活性

60、中心包態(tài)；而且活性中心包含了兩個酸性氨基酸含了兩個酸性氨基酸（一般為谷氨酸和天（一般為谷氨酸和天冬氨酸）冬氨酸）Inverting glycosidase: A single displacement mechanismRetaining glycosidase: A double displacement mechanism親和標記試劑：用于研究特定的活性位點氨基酸殘基。2-脫氧-2-氟糖；5-氟糖。機制：通過鎓離子過渡態(tài)去穩(wěn)定化實現(xiàn)。Mechanism-based, time-dependent inhibitors of retaining glycoside hydrolases.糖苷酶研究工具糖苷酶研究工具Tim