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Letter
Successfully Engineering a Bacterial Sialyltransferase for Regioselective #2,6-sialylation Yangyang Xu, Yueyuan Fan, Jinfeng Ye, Faxing Wang, Quandeng Nie, Li Wang, Peng George Wang, Hongzhi Cao, and Jiansong Cheng ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01993 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018
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ACS Catalysis
Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation Yangyang Xu†, Yueyuan Fan†, Jinfeng Ye‡, Faxing Wang†, Quandeng Nie†, Li Wang†, Peng George Wang*,†, Hongzhi Cao*,‡ and Jiansong Cheng*,† †
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, P. R. China. ‡
National Glycoengineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan 250012, P. R. China. ABSTRACT: A β-galactoside α2,6-sialyltransferase from Photobacterium damsela (Pd2,6ST) that is capable of sialylating both terminal and internal galactose and/or N-acetylgalactosamine was herein redesigned for regioselectively producing terminal α2,6sialylation. Guided by a recently developed bump-hole strategy, a series of mutations at Ala200 and/or Ser232 sites were created for reshaping the acceptor binding pocket. Finally, a Pd2,6ST double mutant A200Y/S232Y with an altered L-shaped acceptor binding pocket was identified to be a superior α2,6-sialyltransferase which can efficiently catalyze the regioselective α2,6sialylation of galactose or N-acetylgalactosamine at the non-reducing end of a series of glycans. Meanwhile, A200Y/S232Y remains flexible donor substrate specificity and is able to transfer Neu5Ac, Neu5Gc and KDN. KEYWORDS： ：sialic acid, α2,6-sialyltransferase, A200Y/S232Y, sialoside, bump-hole
Based on a bump-hole strategy, we herein achieved a SiaT that can only regioslecetively introduce α2,6-linked sialic acid to the Gal or GalNAc terminus of glycan substrates by reshaping the acceptor binding pocket of Pd2,6ST.
INTRODUCTION Sialic acids (Sias) are a family of 9-carbon α-keto acids involving in a broad range of physiologically and pathologically processes.1-6 In human, there are two β-galactoside α2,6-SiaTs (hST6Gal I-II) responsible for the formation of terminal Siaα2,6Gal sequence to various underline glycans. hST6Gal I and hST6Gal II share similar substrate specificities in spite of their different tissue distribution. hST6Gal I is expressed in almost all human tissues, while hST6Gal II is mainly founded in embryonic and adult brain.7-8 The SiaTs of ST6Gal family in mouse have similar substrate specificities as their human counterparts.9
RESULTS AND DISCUSSION Pd2,6ST has been the most widely used biocatalyst to date for preparing α2,6-linked sialosides including α2,6Sialylgangliosides, -N-glycans, -O-glycans, -TACAs (Tumorassociated carbohydrate antigens), and -HMOs (Human milk oligosaccharides), etc.11-12, 15, 19, 22-23 Compared to the rat or human ST6Gal-I, which specially modifies the non-reducing terminal Gal on the Galβ1,4GlcNAc structure and binds the Gal moiety deeply and tightly via a matrix of interactions,24 Pd2,6ST possesses a larger T-shaped acceptor binding pocket that could accommodate internal Gal/GalNAc moieties presented on the glycan acceptors (Figure 1a, Figure S1). Two amino acids (Ala200 and Ser232) located at the bottom of the T-shaped pocket are not conserved within 13 known homogenous SiaTs from the species of Photobacterium. However, all of them bear small side chains (Figure 1c). Recently, a “bumphole” strategy has been developed and proven powerful in generating high-affinity protein-ligand variant pairs,25-26 where the ligand was modified with a sterically bulky substituent that can only be accommodated by the extra space introduced in the pocket of the mutant protein. Herein, we alternatively hypothesized that for the α2,6-SiaT of Pd2,6ST the substitution of Ala200 and/or Ser232 for residues with large side chains would introduce steric clashes between the altered bulky residues and the moiety linked to 3'-oxygen of Gal/GalNAc.
In contrast to mammalian α2,6-SiaTs, those from bacteria, such as Photobacterium damsela Pd2,6ST, usually display more flexible substrate specificity and hence have been widely applied to synthesize sialosides containing various sialic acid forms.10-11 However, in the case of substrates containing multiple Gal/GalNAc residues, such as lacto-N-neotetraose (LNnT)12, galacto-N-biose (GNB)13-14 or the oligo-Nacetyllactosamine (oligo-LacNAc)15-16, Pd2,6ST always catalyzed multiple sialylation and generated heterogeneous sialylation product mixtures. Though mammalian transferases STGal I-II with strict specificity are ideal candidates for synthesis of uniform terminal α2,6-sialylated glycans toward multiGal/GalNAc containing glycoconjugates,17-19 the expression of these recombinant SiaTs in bacteria is often a great challenge. For instance, only 0.27 mgL-1 recombinant hST6Gal I was achieved in Escherichia coli by fusing its functional domain to a maltose-binding protein (MBP).20 Although the expression level was improved to 2 mgL-1 via coexpression of multiple chaperon/foldases in the Origami2 (DE3) strain,21 the amount still does not meet the large-scale synthetic requirements.
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shown in Figure 2, Figure S2 and S3, four double mutants (A200R/S232Q, A200Y/S232R, A200Y/S232Y and A200Q/S232R) did not accept the internal galactose residue of Lc3βproN3 2, while they are still able to efficiently sialylate lactose. Next, the selectivity of these four mutants was further investigated by using Gb3 (Galα1,4Galβ1,4Glc-OH, 3), GM3 (Neu5Acα2,3Galβ1,4Glc-OH, 4) and two O-mannose glycan core structures (Neu5Acα2,3Galβ1,4GlcNAcβ1,6ManαproN3, 5 and Neu5Acα2,3Galβ1,4GlcNAcβ1,2ManαproN3, 6) as acceptor substrates. It is known that α-galactosides are disfavored by the wild type Pd2,6ST.29 Likewise, none of the four mutants was able to recognize Gb3 (Figure 2 and Figure S4). In the case of GM3, the mutants of A200Y/S232R and A200R/S232Q exhibit quite weak activities than A200Q/S232R and the wild type Pd2,6ST, but it is completely not accepted by the A200Y/S232Y mutant (Figure 2 and Figure S4). In addition, the double mutant of A200Y/S232Y demolished the recognition of the SiaT toward two O-mannose glycan core structures (5 and 6) (Figure 2 and Figure S5). According to the predicted model of A200Y/S232Y, the replacement of Ala200 and Ser232 with bumpy tyrosines resulted in a shrunk L-shaped acceptor binding pocket which is consistent with the narrowed regioselectivity of A200Y/S232Y mutant (Figure 1b). Therefore, the A200Y/S232Y mutant would be a functional analogue of hST6Gal for regioselective α2,6-sialylation.
Figure 1. Bump-hole strategy guided mutagenesis of Pd2,6ST. (a) The Tshaped substrate binding pocket of Pd2,6ST is shown in surface representation (PDB ID: 4R84). CMP and lactose were shown as sticks. The lactose binding site on Pd2,6ST was inferred by superposing the Pd2,6ST structure onto another Photobacterium derived α2,6-SiaT Psp26ST (PDB ID: 2Z4T) that has similar global and local structures to Pd2,6ST and was co-crystallized with CMP and lactose. (b) Homology model of the double mutant A200Y/S232Y was generated by using (PS)2 version 3.027. (c) The sequence logo was generated based on 13 homogenous α2,6-SiaTs from Photobacterium with Weblogo (http://weblogo.berkeley.edu/). The residues at positions of 200 and 232 were marked by stars.
According to the above hypothesis, a series of mutations were constructed including six single mutants and nine double mutants, where Ala200 and/or Ser232 were replaced with larger charged (Arg), uncharged (Gln) or aromatic (Tyr) residues. The relative activity and the regioselectivity of individual mutant were screened by using lactose (1) and a trisaccharide Lc3βproN3 (GlcNAcβ1,3Galβ1,4GlcβproN3, 2). As
To assess the catalytic efficiency of A200Y/S232Y mutant, kinetics parameters were determined by using fluorescent 4methylumbelliferyl-β-D-lactoside (LacβMU) as acceptor substrate and fitting the data into the Michaelis–Menten equation using Grafit 6.0 (http://www.erithacus.com/grafit/). Compared to the wild type Pd2,6ST, the engineered mutant (A200Y/S232Y) has comparable catalytic rate constant against both CMP-Neu5Ac and LacβMU in spite of increased Km values (Table 1). Since Ala200 and Ser232 locate beyond the donor binding pocket, replacement of them with Tyr did not alter the donor specificity of the enzyme (A200Y/S232Y can transfer three natural occurring Sias including KDN, Neu5Ac and Neu5Gc, Figure S6). Additionally, the hydrolysis efficiency (kcat/Km) of A200Y/S232 for CMP-Neu5Ac in absence of acceptor is very close to that of wild type Pd2,6ST (9.0 mM1 ·min-1 versus 8.8 mM-1·min-1) (Table S2). Moreover, the expression level of A200Y/S232Y (32 mgL−1 culture) is very close to that of the wild type enzyme (36 mgL−1 culture). The double mutant of A200Y/S232Y was then employed for preparative synthesis of seven terminal α2,6-sialylated natural or unnatural sialosides in a one-pot multi-enzyme manner. Table 1. Apparent Kinetic Parameters of Pd2,6ST and the A200Y/S232Y Mutant. Enzymes Wild type Pd2,6ST Substrates CMP-Neu5Ac LacβMU Km (mM) 1.4±0.2 0.4±0.05 Vmax (µM·min-1) 4.6±0.4 2.7±0.1 kcat (S-1) 1.5 0.9 kcat/Km (mM-1·S-1) 1.1 2.0
Mutant A200Y/S232Y CMP-Neu5Ac LacβMU 2.7±0.4 1.5±0.06 4.7±0.4 11.8±0.2 1.5 1.1 0.6 0.7
Galacto-N-biose (GNB, Galβ1,3GalNAc) is a common disaccharide epitope involved in many biological events, such as cell adhesion, cancer metastasis, etc. GNB with an α configuration (Galβ1,3GalNAcα-O-Ser/Thr) so-called ThomsenFriedenreich antigen (TF or T-antigen) is commonly overexpressed on human cancer cells.30-31 GNB with β configuration (Galβ1,3GalNAcβ) instead is an essential component of globo/ganglio-series glycosphingolipids, such as Globo-H, Gb5, GD1aα, GT1aα, etc.32-33 Herein, the disaccharide GNBβproN3
Figure 2. Acceptor substrates specificities of Pd2,6ST and its mutants determined by TLC analysis. All assays were carried out in a volume of 50 uL containing 10 mM (1.0 equiv.) of individual acceptor and 1.5 equiv. of Neu5Ac and CTP, which were in situ converted to CMP-Neu5Ac by NmCSS (CMP–sialic acid synthetase from Neisseria meningitidis group B28). aMono-sialylated compound found. bNot detected. cNot tested. d Mono-sialylated compound was detected with low catalytic efficiency.
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ACS Catalysis mon structures of human milk oligosaccharides (HMOs),35 which are a family of unconjugated glycans being unique to human milk and benefit the breast-fed infants in multiple ways.36 As shown in Figure 4, LNnTβproN3 (15) is able to be facilely converted to LSTcβproN3 (16) by A200Y/S232Y in a high yield (90%).
(7) and GNBαproN3 (8) were prepared in a one-pot multienzyme system as described previously.34 We have demonstrated that the sialylation of Galβ1,3GalNAcβproN3 (7) catalyzed by the wild type Pd2,6ST always yield a mixture of three sialosides (sialylated on Gal, GalNAc and both of them) with a certain ratio (3:3:1) when 1.0 equiv. of Neu5Ac and CTP were afforded in a one-pot multienzyme system.13 In the present study, a sensitive and quantitative method of HILICELSD (hydrophilic interaction liquid chromatography with evaporative light scattering detection) was developed for monitoring the sialylation reactions of disaccharide 7 catalyzed by Pd2,6ST and the A200Y/S232Y mutant. When 0.5 equiv. of CMP-Neu5Ac was used for the Pd2,6ST-catalyzed reaction, the yields of monosialyl trisaccharide 9 with Neu5Acα2,6linked to the terminal Gal, monosialyl trisaccharide 10 with Neu5Acα2,6-linked to the internal GalNAc, and disialyl tetrasaccharide 11 were 13.7%, 9.8%, and 10.3%, respectively. With the amount of CMP-Neu5Ac increased (1.5 and 3.0 equiv.), disialyl tetrasaccharide 11 turned into the dominated product (Figure 3 and Figure S7-S9). In contrast, A200Y/S232Y-catalyzed reaction for disaccharide 7 yielded uniform terminal α2,6-sialylated product of Neu5Acα2,6Galβ1,3GalNAcβproN3. Furthermore, KDN/Neu5Gcα2,6Galβ1,3GalNAcβproN3 and terminal α2,6sialyl Galβ1,3GalNAcαproN3 was also prepared by using A200Y/S232Y (Figure 4).
Oligo-N-acetyllactosamine (oligo-LacNAc) chains are a common substructures found on various N- and O-linked glycans.37 The wild type Pd2,6ST has been demonstrated to modify both the terminal and internal galactose residues, resulting sialylated mixtures.15-16 The regioselectivity of A200Y/S232Y mutant for the di-LacNAc containing hexasaccharide (paraLacto-N-neohexaose, para-LNnH, 17) was further investigated. As expected, the reaction catalyzed by the mutant gave only terminal sialylated product 19 (Neu5Acα2,6Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Galβ1,4 GlcβproN3). In addition, GM2 is also a good substrate for A200Y/S232Y mutant to provide terminal α2,6-sialyl GM2 (Neu5Acα2,6GalNAcβ1,4(Neu5Acα2,3)Galβ1,4GlcβOMe, 20). The reaction process for acceptors 7-8, 15, 17-18 was tracked and analyzed by using time-course HILIC-ELSD prior to scaling up the reaction in a one-pot multi-enzyme manner (Figure S8, S10, S12, S14, S16). For 7, 15 and 17, the wild type Pd2,6ST-catalyzed multiple sialylation and generated a mixture of mono-, di- and trisialylated (for 17) products. However, terminal α2,6-sialylation of all five acceptors was exclusively achieved by the A200Y/S232Y mutant. Interestingly, GM2 (18) was also sialylated to the terminal GalNAc by Pd2,6ST possibly due to a steric clash between the β1,4linked GalNAc and the naturally occurring residue that blocks the entrance of the internal Gal moiety (Figure 1a). Nuclear magnetic resonance (NMR) data confirmed that the attachment of Neu5Ac in the mono-sialylated product was to the C-6 of the terminal Gal or GalNAc residue in all A200Y/S232Y catalyzed reactions. Recently, by engineering amino acids at two conserved sites (P7H and M117A in Pd2,3ST, and P34H and M144L in PmST1), two bacterial α2,3-SiaTs that catalyze the terminal α2,3-sialylation were converted to α2,6-selective transferases (the formation of α2,6-sialyl linkage versus α2,3-sialyl linkage was changed to over 99.5% and 98.7% for Pd2,3ST and PmST1, respectively.38-39) However, when the PmST1 P34H/M144L mutant was applied for the synthesis of Neu5Acα2,6-LNnT in presence of 1.0 equiv. of CMP-Neu5Ac, 5% of the disialylated by-product was still produced. It implied that the regioselectivity of the P34H/M144L mutant toward terminal Gal/GalNAc was also altered with the switch of linkage specificity mainly from α2,3 to α2,6. In this study, we alternatively achieved a SiaT that can regioselectively produce terminal α2,6-sialylation guided by the bump-hole strategy.
Figure 3. Time-course studies of Pd2,6ST (Green) and A200Y/S232Y (Red) -catalyzed α2,6-sialylation of disaccharide 7 with various amount of CMP-Neu5Ac (0.5/1.5/3.0 equiv.) afforded. All reactions were incubated at 37 °C for 4 h.
Lacto-N-neotetraose (LNnT, Galβ1,4GlcNAcβ1,3Galβ1,4Glc) and its terminal mono-sialyl derivative of LSTc (Neu5Acα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glc) are two com-
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OH -
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20 (80%)
Figure 4. Enzymatic synthesis of terminal α2,6-linked sialosides by using the engineered mutant of A200Y/S232Y. Reagents: Tris-HCl (100 mM, pH 8.5), Individual acceptor (10 mM, 1.0 equiv.), MgCl2 (20 mM), CTP (1.5 equiv.), NmCSS28 and A200Y/S232Y; (i) Neu5Ac (1.5 equiv.); (ii) Mannose (1.5 equiv.), sodium pyruvate (7.5 equiv.), E. coli aldolase28; (iii) ManNGc (1.5 equiv.), sodium pyruvate (7.5 equiv.), E. coli aldolase.
Taken together, a widely used bacterial α2,6-SiaT (Pd2,6ST) was herein redesigned to be a functional analogue of mammalian ST6Gal I. In contrast to ST6Gal I, which catalyzes the α2,6-sialylation of the terminal Gal with Galβ1,4GlcNAc as the preferred structure, the engineered A200Y/S232Y mutant is capable of regioselectively sialylating both Gal and GalNAc at the non-reducing end of various glycans. Further, the transferase activity of A200Y/S232Y toward terminal GalNAc differs from that of hST6GalNAc family, since hST6GalNAc I and II recognize both terminal and internal GalNAc on Tn antigen (GalNAcα-O-Ser/Thr), T antigen (Galβ1,3GalNAcα-O-Ser/Thr) and the 3'-sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα-O-Ser/Thr),40 while hST6GalNAc III-VI exclusively sialylate the internal GalNAc residue. Therefore, the A200Y/S232Y mutant presents a new superior α2,6-SiaT for highly efficient synthesis of various Siaα2,6Gal- and Siaα2,6GalNAc-linked glycans for further indepth biological studies. Meanwhile, the bump-hole strategy demonstrated valuable for tailoring the regioselectivity of SiaTs especially for those having high specific activity and flexible substrate specificity.
*E-mail for Hongzhi Cao:
[email protected], *E-mail for Peng George Wang:
[email protected] Notes The authors declare no competing financial interest.
ASSOCIATED CONTENT Supporting Information Experimental details and characteration of products. The Supporting information is available free of charge on the ACS Publications website (http://pubs.acs.org).
ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21372130 and 81302682).
REFERENCES (1) Varki, N. M.; Varki, A. Diversity in Cell Surface Sialic Acid Presentations: Implications for Biology and Disease. Lab. Invest. 2007, 87, 851-857. (2) Varki, A. Sialic Acids in Human Health and Disease. Trends Mol. Med. 2008, 14, 351-360. (3) Schnaar, R. L.; Gerardy-Schahn, R.; Hildebrandt, H. Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration. Physiol. Rev. 2014, 94, 461-518.
AUTHOR INFORMATION Corresponding Author *E-mail for Jiansong Cheng:
[email protected],
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(4) Varki, A. Glycan-Based Interactions Involving Vertebrate Sialic-acid-Recognizing Proteins. Nature 2007, 446, 10231029. (5) Xiong, X.; Martin, S. R.; Haire, L. F.; Wharton, S. A.; Daniels, R. S.; Bennett, M. S.; McCauley, J. W.; Collins, P. J.; Walker, P. A.; Skehel, J. J. Receptor Binding by an H7N9 Influenza Virus from Humans. Nature 2013, 499, 496-499. (6) Büll, C.; Stoel, M. A.; den Brok, M. H.; Adema, G. J. Sialic Acids Sweeten a Tumor's Life. Cancer Res. 2014, 74, 31993204. (7) Takashima, S.; Tsuji, S.; Tsujimoto, M. Characterization of the Second Type of Human β-Galactoside α2,6Sialyltransferase (ST6Gal II), which Sialylates Galβ1,4GlcNAc Structures on Oligosaccharides Preferentially. J. Biol. Chem. 2002, 277, 45719-45728. (8) Krzewinski-Recchi, M.-A.; Julien, S.; Juliant, S.; Teintenier-Lelièvre, M.; Samyn-Petit, B.; Montiel, M.-D.; Mir, A.-M.; Cerutti, M.; Harduin-Lepers, A.; Delannoy, P. Identification and Functional Expression of a Second Human β-Galactoside α2,6-Sialyltransferase, ST6Gal II. Eur. J. Biochem. 2003, 270, 950-961. (9) Takashima, S. Characterization of Mouse Sialyltransferase Genes: Their Evolution and Diversity. Biosci. Biotechnol. Biochem. 2008, 72, 1155-1167. (10) Yu, H.; Chokhawala, H. A.; Huang, S.; Chen, X. One-Pot Three-Enzyme Chemoenzymatic Approach to the Synthesis of Sialosides Containing Natural and Non-Natural Functionalities. Nat. Protoc. 2006, 1, 2485-2492. (11) Yu, H.; Huang, S.; Chokhawala, H.; Sun, M.; Zheng, H.; Chen, X. Highly Efficient Chemoenzymatic Synthesis of Naturally Occurring and Non-Natural α-2,6-Linked Sialosides: A P. damsela α-2,6-Sialyltransferase with Extremely Flexible Donor–Substrate Specificity. Angew. Chem. Int. Ed. 2006, 45, 3938-3944. (12) Yu, H.; Lau, K.; Thon, V.; Autran, C. A.; Jantscher‐ Krenn, E.; Xue, M.; Li, Y.; Sugiarto, G.; Qu, J.; Mu, S.; Ding, L.; Bode, L.; Chen, X. Synthetic Disialyl Hexasaccharides Protect Neonatal Rats from Necrotizing Enterocolitis. Angew. Chem. Int. Ed. 2014, 53, 6687-6691. (13) Meng, X.; Yao, W.; Cheng, J.; Zhang, X.; Jin, L.; Yu, H.; Chen, X.; Wang, F.; Cao, H. Regioselective Chemoenzymatic Synthesis of Ganglioside Disialyl Tetrasaccharide Epitopes. J. Am. Chem. Soc. 2014, 136, 5205-5208. (14) Yu, H.; Yan, X.; Autran, C. A.; Li, Y.; Etzold, S.; Latasiewicz, J.; Robertson, B. M.; Li, J.; Bode, L.; Chen, X. Enzymatic and Chemoenzymatic Syntheses of Disialyl Glycans and Their Necrotizing Enterocolitis Preventing Effects. J. Org. Chem. 2017, 82, 13152-13160. (15) Nycholat, C. M.; Peng, W.; McBride, R.; Antonopoulos, A.; de Vries, R. P.; Polonskaya, Z.; Finn, M.; Dell, A.; Haslam, S. M.; Paulson, J. C. Synthesis of Biologically Active Nand O-Linked Glycans with Multisialylated Poly-Nacetyllactosamine Extensions Using P. damsela α2-6 Sialyltransferase. J. Am. Chem. Soc. 2013, 135, 18280-18283. (16) Chien, W. T.; Liang, C. F.; Yu, C. C.; Lin, C. H.; Li, S. P.; Primadona, I.; Chen, Y. J.; Mong, K. K.; Lin, C. C. Sequential One-Pot Enzymatic Synthesis of Oligo-Nacetyllactosamine and Its Multi-Sialylated Extensions. Chem. Commun. 2014, 50, 5786-5789. (17) Nycholat, C. M.; McBride, R.; Ekiert, D. C.; Xu, R.; Rangarajan, J.; Peng, W.; Razi, N.; Gilbert, M.; Wakarchuk, W.; Wilson, I. A.; Paulson, J. C. Recognition of Sialylated Poly-N-acetyllactosamine Chains on N- and O-Linked Glycans by Human and Avian Influenza A Virus Hemagglutinins. Angew. Chem. Int. Ed. 2012, 51, 4860-4863.
(18) Prudden, A. R.; Liu, L.; Capicciotti, C. J.; Wolfert, M. A.; Wang, S.; Gao, Z.; Meng, L.; Moremen, K. W.; Boons, G.-J. Synthesis of Asymmetrical Multiantennary Human Milk Oligosaccharides. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 69546959. (19) Peng, W.; Paulson, J. C. CD22 Ligands on a Natural NGlycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells. J. Am. Chem. Soc. 2017, 139, 12450-12458. (20) Hidari, K. I.; Horie, N.; Murata, T.; Miyamoto, D.; Suzuki, T.; Usui, T.; Suzuki, Y. Purification and Characterization of a Soluble Recombinant Human ST6Gal I Functionally Expressed in Escherichia coli. Glycoconj. J. 2005, 22, 1-11. (21) Ortiz-Soto, M. E.; Seibel, J. Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli. PloS One 2016, 11, e0155410. (22) Wang, Z.; Gilbert, M.; Eguchi, H.; Yu, H.; Cheng, J.; Muthana, S.; Zhou, L.; Wang, P. G.; Chen, X.; Huang, X. Chemoenzymatic Syntheses of Tumor-Associated Carbohydrate Antigen Globo-H and Stage-Specific Embryonic Antigen 4. Adv. Synth. Catal. 2008, 350, 1717-1728. (23) Li, L.; Liu, Y.; Li, T.; Wang, W.; Yu, Z.; Ma, C.; Qu, J.; Zhao, W.; Chen, X.; Wang, P. G. Efficient Chemoenzymatic Synthesis of Novel Galacto-N-Biose Derivatives and Their Sialylated Forms. Chem. Commun. 2015, 51, 10310-10313. (24) Meng, L.; Forouhar, F.; Thieker, D.; Gao, Z.; Ramiah, A.; Moniz, H.; Xiang, Y.; Seetharaman, J.; Milaninia, S.; Su, M. Enzymatic Basis for N-Glycan Sialylation: Structure of Rat α2,6-Sialyltransferase (ST6GAL1) Reveals Conserved and Unique Features for Glycan Sialylation. J. Biol. Chem. 2013, 288, 34680-34698. (25) Islam, K.; Chen, Y.; Wu, H.; Bothwell, I. R.; Blum, G. J.; Zeng, H.; Dong, A.; Zheng, W.; Min, J.; Deng, H. Defining Efficient Enzyme–Cofactor Pairs for Bioorthogonal Profiling of Protein Methylation. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 16778-16783. (26) Baud, M. G.; Lin-Shiao, E.; Cardote, T.; Tallant, C.; Pschibul, A.; Chan, K.-H.; Zengerle, M.; Garcia, J. R.; Kwan, T. T.-L.; Ferguson, F. M. A Bump-and-Hole Approach to Engineer Controlled Selectivity of BET Bromodomain Chemical Probes. Science 2014, 346, 638-641. (27) Chen, C.-C.; Hwang, J.-K.; Yang, J.-M. (PS)2: Protein Structure Prediction Server. Nucleic Acids Res. 2006, 34, W152-W157. (28) Yu, H.; Yu, H.; Karpel, R.; Chen, X. Chemoenzymatic Synthesis of CMP-Sialic Acid Derivatives by a One-Pot TwoEnzyme System: Comparison of Substrate Flexibility of Three Microbial CMP-Sialic Acid Synthetases. Bioorg. Med. Chem. 2004, 12, 6427-6435. (29) Yamamoto, T.; Nakashizuka, M.; Kodama, H.; Kajihara, Y.; Terada, I. Purification and Characterization of a Marine Bacterial β-Galactoside α2,6-Sialyltransferase from Photobacterium damsela JTO16O. J. Biochem. 1996, 120, 104-110. (30) Hanisch, F.-G. O-Glycosylation of the Mucin Type. Biol. Chem. 2001, 382, 143-149. (31) Dube, D. H.; Bertozzi, C. R. Glycans in Cancer and Inflammation–Potential for Ttherapeutics and Diagnostics. Nat. Rev. Drug Discov. 2005, 4, 477-488. (32) Todeschini, A. R.; Hakomori, S. I. Functional Role of Glycosphingolipids and Gangliosides in Control of Cell Adhesion, Motility, and Growth, through Glycosynaptic Microdomains. Biochim. Biophys. Acta. 2008, 1780, 421-433. (33) Lingwood, C. A. Glycosphingolipid Functions. Cold Spring Harbor Perspect. Biol. 2011, 3, a004788. (34) Yu, H.; Thon, V.; Lau, K.; Cai, L.; Chen, Y.; Mu, S.; Li, Y.; Wang, P. G.; Chen, X. Highly Efficient Chemoenzymatic
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Synthesis of β1–3-Linked Galactosides. Chem. Commun. 2010, 46, 7507-7509. (35) Chen, X. Chapter Four-Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis. Adv. Carbohydr. Chem. Biochem. 2015, 72, 113-190. (36) Bode, L. Human Milk Oligosaccharides: Every Baby Needs a Sugar Mama. Glycobiology 2012, 22, 1147-1162. (37) Ujita, M.; Misra, A. K.; McAuliffe, J.; Hindsgaul, O.; Fukuda, M. Poly-N-acetyllactosamine Extension in N-Glycans and Core 2- and Core 4-branched O-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family. J. Biol. Chem. 2000, 275, 15868-15875. (38) Schmölzer, K.; Czabany, T.; Luley-Goedl, C.; PavkovKeller, T.; Ribitsch, D.; Schwab, H.; Gruber, K.; Weber, H.;
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Nidetzky, B. Complete Switch from α–2,3- to α–2,6Regioselectivity in Pasteurella dagmatis β-D-Galactoside Sialyltransferase by Active-Site Redesign. Chem. Commun. 2015, 51, 3083-3086. (39) McArthur, J. B.; Yu, H.; Zeng, J.; Chen, X. Converting Pasteurella multocida α2–3-Sialyltransferase 1 (PmST1) to a Regioselective α2–6-Sialyltransferase by Saturation Mutagenesis and Regioselective Screening. Org. Biomol. Chem. 2017, 15, 1700-1709. (40) Marcos, N. T.; Pinho, S.; Grandela, C.; Cruz, A.; SamynPetit, B.; Harduin-Lepers, A.; Almeida, R.; Silva, F.; Morais, V.; Costa, J. Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen. Cancer Res. 2004, 64, 7050-7057.
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Figure 1. Bump-hole strategy guided mutagenesis of Pd2,6ST. (a) The T-shaped substrate binding pocket of Pd2,6ST is shown in surface representation (PDB ID: 4R84). CMP and lactose were shown as sticks. The lactose binding site on Pd2,6ST was inferred by superposing the Pd2,6ST structure onto another Photobacterium derived α2,6-SiaT Psp26ST (PDB ID: 2Z4T) that has similar global and local structures to Pd2,6ST and was co-crystallized with CMP and lactose. (b) Homology model of the double mutant A200Y/S232Y was generated by using (PS)2 version 3.027. (c) The sequence logo was generated based on 13 homogenous α2,6-SiaTs from Photobacterium with Weblogo (http://weblogo.berkeley.edu/). The residues at positions of 200 and 232 were marked by stars. 83x53mm (300 x 300 DPI)
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Figure 2. Acceptor substrates specificities of Pd2,6ST and its mutants determined by TLC analysis. All assays were carried out in a volume of 50 uL containing 10 mM (1.0 equiv.) of individual acceptor and 1.5 equiv. of Neu5Ac and CTP, which were in situ converted to CMP-Neu5Ac by NmCSS (CMP–sialic acid synthetase from Neisseria meningitidis group B28). aMonosialylated compound found. bNot detected. cNot tested. dMonosialylated compound was detected with low catalytic efficiency. 110x133mm (300 x 300 DPI)
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Figure 3. Time-course studies of Pd2,6ST (Green) and A200Y/S232Y (Red)-catalyzed α2,6-sialylation of disaccharide 7 with various amount of CMP-Neu5Ac (0.5/1.5/3.0 equiv.) afforded. All reactions were incubated at 37 °C for 4 h. 73x51mm (300 x 300 DPI)
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Figure 4. Enzymatic synthesis of terminal α2,6-linked sialosides by using the engineered mutant of A200Y/S232Y. Reagents: Tris-HCl (100 mM, pH 8.5), Individual acceptor (10 mM, 1.0 equiv.), MgCl2 (20 mM), CTP (1.5 equiv.), NmCSS 28 and A200Y/S232Y; (i) Neu5Ac (1.5 equiv.); (ii) Mannose (1.5 equiv.), sodium pyruvate (7.5 equiv.), E. coli aldolase 28; (iii) ManNGc (1.5 equiv.), sodium pyruvate (7.5 equiv.), E. coli aldolase. 517x420mm (300 x 300 DPI)
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