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An Effective Bacterial Fucosidase for Glycoprotein Remodeling Tsung-I Tsai, Shiou-Ting Li, Chiu-Ping Liu, Karen Y. Chen, Sachin S. Shivatare, Chin-Wei Lin, Shih-Fen Liao, Chih-Wei Lin, Tsui-Ling Hsu, Ying-Ta Wu, MingHung Tsai, Meng-Yu Lai, Nan-Horng Lin, Chung-Yi Wu, and Chi-Huey Wong ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00821 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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ACS Chemical Biology

An Effective Bacterial Fucosidase for Glycoprotein Remodeling

Tsung-I Tsai1,2,3, Shiou-Ting Li1, Chiu-Ping Liu1,2, Karen Y. Chen1,4, Sachin S. Shivatare1, Chin-Wei Lin1, Shih-Fen Liao1, Chih-Wei Lin1, Tsui-Ling Hsu1, Ying-Ta Wu1, Ming-Hung Tsai5, Meng-Yu Lai5, Nan-Horng Lin5, Chung-Yi Wu1, Chi-Huey Wong1,2,3*

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Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan

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Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan

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Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines

Road, La Jolla, CA 92037 4

Department of Chemistry, University of Michigan, Ann Arbor, MI 48109

5

CHO Pharma Inc., Taipei 11503, Taiwan

KEYWORDS: Fucosidase, remodeling, homogeneous glycoprotein, core fucose, glycan sequencing

E-mail of corresponding author: [email protected]

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ABSTRACT Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-L-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endo-glycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.

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INTRODUCTION Protein glycosylation is a co- or post-translational process that proceeds through a series of steps, including addition and trimming of glycans with glycosyltransferases and glycosidases, respectively. These modifications occur without a template and result in a heterogeneous population of glycoproteins1 and as such, it has been difficult to prepare homogeneous glycoproteins with well-defined glycan structures to study the effect of glycosylation on the structure and function of glycoproteins. Currently, most glycoproteins under development are monoclonal antibodies (such as Humira, Herceptin and Rituxan), although other glycoproteins such as erythropoietin (EPO), clotting factors (Factor VIII), cytokines (Interferon), enzymes (Idursulfase), enzyme inhibitors (Antithrombin), and Fc-fusion proteins (Fc-TNF receptor fusion protein) have also been developed. However, all these glycoprotein pharmaceuticals have been manufactured as mixtures of glycoforms, though the glycan chains on glycoproteins are known to affect their folding, structure, bioactivity, and a number of other properties, such as trafficking, solubility, stability against proteolysis, immunogenicity, and clearance.2-4 Most therapeutic antibodies, for example, have a glycan with heterogeneous compositions attached to the Asn-297 residue in the Fc region, capped with mannose (Man), N-acetylglucosamine (GlcNAc), galactose (Gal) or neuraminic acid (NeuAc) in the α-2,3/6 linkage or with fucose (Fuc) in the α-1,2 linkage to the Gal residue, or the α-1,3 or 1,4 linkage to the outer GlcNAc residue, or the α−1,6 linkage to the innermost GlcNAc residue (the core fucose). These N-glycan compositions dramatically influence the IgG binding to the Fcγ receptors (FcγRs) and C1q receptors on immune cells, and thus affect antibody activities (the effector functions).5-11 Several studies have demonstrated that antibodies without core fucose have much higher avidity for FcγRIIIa than the core fucosylated antibody,12, 13 resulting in an enhancement of antibody-dependent cellular cytotoxicity (ADCC).14, 15 Therefore, many strategies have been devised to improve the IgG-FcγR interaction to enhance the effector functions, and among the strategies, engineering of the Fc-glycans to eliminate the core fucose has been noted as a key modification for the preparation of therapeutic antibodies with enhanced efficacy.14, 16, 17 Further studies have shown the effect of terminal glycosylation, especially 2,3- vs 2,6-sialylation, on the effector functions11. Of numerous methods directed towards the preparation of antibodies without core fucose, the most common one is based on the modification of the fucosylation pathway in mammalian cell lines, including: (1) knock-out/down of α-1,6-fucosyltransferase, which is responsible for the transfer of fucose from GDP-fucose to the innermost GlcNAc of the tri-mannosyl core structure capped with GlcNAc18, 19; (2) 3



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knock-out/down of one or more enzymes involved in the synthesis of GDP-fucose including the enzymes in the de novo or the salvage pathway to produce defucosylated IgG;16, 19, 20 and (3) small molecule-based inhibitors of cellular protein fucosylation. Recent two studies have shown that fluorinated fucose analogs can be taken up and metabolized to the GDP derivatives, which either inhibit the fucosyltransferase family activity21 or block the key enzyme, GDP-mannose 4,6-dehydratase, in the de novo pathway of GDP-fucose formation.22 However, none of the pathway engineering methods is able to provide homogenous glycoproteins for use to study the effect of glycosylation on the structure and function of glycoproteins and to develop homogeneous glycoproteins with well-defined glycan structure and optimized activity. The N-glycans found on human glycoproteins are estimated to exceed 20,000 sequences23 and enzymatic fucosylations contribute most significantly to the structural diversity of N-glycans which are difficult to distinguish by the current mass spectrometry method. In addition, there is lack of specific fucosidase available for each of the fucosidic linkages, especially for the α-1,6-linked core fucose. So far, only one report has shown the hydrolysis of core fucose using a nonspecific α-L-fucosidase from bovine kidney; however, the method requires a relatively large amount of α-L-fucosidase and a prolonged reaction time to achieve a complete defucosylation.9 Therefore, development of effective fucosidases for use to selectively remove the 1,6-linked fucose as well as other linkages would facilitate the understanding of glycosylation in biology and the development of pharmaceutical glycoproteins. In this study, we report a bacterial fucosidase, which is capable of efficient removal of the core fucose and other fucosidic linkages from various glycans, glycocopeptides and glycoproteins at neutral pH with some selectivity, and is useful for the preparation of homogeneous glycoproteins with well-defined glycan structures (Scheme 1).

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RESULTS AND DISCUSSION Preparation of a library of bacterial fucosidases for screen Many bacteria express high levels of α-L-fucosidases that prefer acidic conditions, and some have broad substrate specificities.24-26 However, none of these enzymes has the desired property for use to remodel the fucose-containing glycans of antibodies. In order to identify the fucosidases with broad hydrolysis activity under neutral conditions, a panel of bacterial fucosidase genes from the CAZy glycoside hydrolase (GH) family 29 (mainly α-L-fucosidases)27 was cloned and expressed in E. coli. The substrate specificity of recombinant fucosidases was first screened by use of relatively small fucosyl-conjugates, including various p-nitrophenyl (pNP) glycosides, fucosyllactoses, fucose-containing tumor-associated carbohydrate antigens (TACAs), and Lewis as well as sialyl-Lewis antigens. The fucosidase activity was identified by mixing one of these fucosylated glycans with recombinant enzymes followed by thin-layer chromatography (TLC) of the reaction mixture. Of many fucosidases from the library tested (Supplementary Table S2), the one designated as BfFucH (BF3242 in the genome of Bacteroides fragilis NCTC 9343) was found to hydrolyze the α-fucosidic bond of the 1,6 and various linkages (Supplementary Figure S1 and S2). The cleavage of the α-1,6-linked core fucose from GlcNAc was screened with the use of glycan A2F and its derivatives. This enzyme exhibited an increase in the cleavage activity toward core fucose with decrease in glycan length. Among the glycans with core fucose tested, the trisaccharide M1N2F is the best substrate with a 53.3% digestion rate. For further quantitative analysis of the substrate specificity of BfFucH, we used the 2-AB tagged glycans with different fucose linkages as substrates and the digestion products were analyzed by UPLC (Table 1). A complete removal of terminal α-1,3 and α-1,4-linked fucose from the N-acetylglucosamine (GlcNAc) residue linked to the α-1,3 or the α-1,6 mannose arm of the N-glycans by BfFucH was observed. However, short glycans with fucose α-1,3/4-linked to GlcNAc (LNFP III, and LN FP II) have very little cleavage activity (less than 5%), whereas the α-1,2 linkage to the galacose residue (LNFP I) was cleaved efficiently. Among the Lewis (Le) blood group antigens, Lea, Leb, Lex, Ley, and sialyl Lex (sLex), only Ley, which has an α-1,2-linked fucose on the galactose and an α-1,3 linkage to the GlcNAc, showed a complete cleavage of the α-1,2 linkage, while Leb, which has an α-1,2-linked fucose on the galactose in addition to the α-1,4-linked fucose on the GlcNAc, showed no cleavage activity at all, suggesting that the α-1,4-, but not the α-1,3-linked fucose could block BfFucH from cleaving the α-1,2-linked fucose on galactose. Lea and Lex, the glycans with the fucose residue linked to the GlcNAc residue that are hindered by a terminal galactose, showed 5



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no digestion either. Hydrolysis of sLex was also attempted, but no digestion was observed. On the other hand, we observed a complete digestion of the α-1,2-linked fucose on Globo H, a glycan used as epitope for the synthesis of an anticancer vaccine.28, 29 In summary, our study showed that BfFucH can specifically hydrolyze the core fucose linkage of N-linked glycans, especially with an appreciable digestion rate toward the N-glycans with terminal sialylation. The enzyme can also cleave the α-1,2-linked fucose on galactose and α-1,3/4-linked fucose on GlcNAc when the GlcNAc is not capped with galactose. The activity of BfFucH was next evaluated at different pH ranges. When compared to other fucosidases, which have optimal activities under acidic conditions (pH 4.0-6.0),30-38 BfFucH functions best under milder conditions (pH 7.0-7.5) (Figure 1a). Also, it is not affected by most common divalent metal ions, except Ni2+, which dramatically reduced the enzymatic activity to 60%, whereas Zn2+ and Cu2+ completely abolished the activity. However, the chelator EDTA has no influence on the enzyme activity, indicating that the metal ion does not participate in the catalysis (Figure 1b). Furthermore, BfFucH exhibited a typical Arrhenius behavior with moderate activity at room temperature compared to high temperature (60°C), and still had 20% relative activity even at 4°C. The activity at low temperature may be desirable for the treatment of some macromolecules due to their intrinsic instability or heat-labile property (Figure 1c). In addition, BfFucH showed a comparable or even higher activity when compared with other reported fucosidases (Supplementary Table S3). The high specificity and broad substrate tolerance of BfFucH encouraged us to exploit its use for the removal of fucose from glycans and glycoproteins (Figure 1d). Cleavage of core fucose in IgG We examined the cleavage activity of BfFucH on core fucose using the commercially available antibody Rituximab as substrate. The N-glycans in the IgG Fc domain of Rituximab are complex-type biantennary structures containing 0, 1, or 2 terminal galactoses corresponding to G0, G1, and G2 glycoforms, respectively. Less than 10% of the N-glycan structures are sialylated, but more than 90% are fucosylated in the core region. Our preliminary study showed that BfFucH is relatively inactive toward the core region of intact glycoprotein. However, BfFucH showed very high defucosylation activity in the presence of endoS,39 which is an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, and can cleave the glycan chain to leave the innermost GlcNAc of N-glycans. In a recent study, Wang et al. used relatively large amounts of α-L-fucosidase from bovine kidney at low pH (pH 5.5)9 and from Lactobacillus casei (after submission of this paper) at mild pH conditions40 to remove the core fucose. In 6


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our comparative study based on Aleuria aurantia lectin (AAL) blotting (Figure 2), BfFucH is much more active for the defucosylation of GlcNAc-Fuc antibody, giving the mono-GlcNAc product in 97% yield in 8 hours, compared to 71% using the recombinant Lactobacillus casei α-L-fucosidases and 2% using the commercially available bovine α-L-fucosidase (Figure 2a, 2b and 2c).9 Two other well-studied fucosidases from Bacteroides thetaiotaomicron38 (Figure 2d) and Thermotoga maritima30 (Figure 2e) were also included for comparison, and the result showed that BfFucH has the highest activity for the cleavage of core fucose. Moreover, a time course study was carried out using the endoS-treated Rituxan as substrate at different temperatures (Figure 3). The core fucose was completely hydrolyzed after 12 h at 37 °C, or 24 h at 30 °C. This result demonstrates that BfFucH-catalyzed defucosylation reaction can be performed at low temperature with shorter reaction time than the enzyme from bovine kidney and Lactobacillus, and it has been used in our laboratory for the preparation of mono-GlcNAc proteins for further glycan elongation and functional studies.11

Use of BfFucH to remove core fucose in other glycoproteins The core fucosylation not only influences the interaction between IgG and FcγR, but is also involved in other biological processes, such as dysregulation of transforming growth factor-β1 (TGF-β1) receptor activation41 and regulation of non-small cell lung cancer progression.42 However, the role of core fucose in other glycoproteins remains unclear. Thus, our next focus was to investigate the specificity and activity of BfFucH toward other glycoproteins, including the monoclonal antibody Humira (fully human IgG1 monoclonal antibody (mAb)), tumor necrosis factor receptor (TNFR)-Fc fusion protein (Enbrel), erythropoietin (EPO1β), interferon (IFNβ1a), and the influenza glycoprotein hemagglutinin (HA). To evaluate the enzymatic activity of BfFucH on these glycoproteins, the recombinant BfFucH was incubated with a glycoprotein together with a cocktail of endoglycosidases (endoF1, F2, F3, H and S) and the product was analyzed by SDS-PAGE and AAL lectin blotting (Figure 4). The glycoprotein sample treated with PNGase F (lane 4, in the bottom of figure) or with the endoglycosidases cocktail and BfFucH (line 3) showed no signal, whereas the samples treated with the cocktail of endoglycosidases (line 2) to expose the core fucose, display a strong signal in AAL lectin blotting. This result indicated that all of the tested glycoproteins containing core fucose are substrates for BfFucH.

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The most significant result was observed in the study of EPO1β, a highly glycosylated glycoprotein with three intrinsic N-glycosites and one O-glycosite. In general, these glycosylation sites carry multi-antennary N-glycans, which are not easily hydrolyzed by the cocktail of endo-glycosidases. But in this study, a ladder-like signal was seen in the Western blot, which was not observed in the AAL lectin blotting, probably due to the high de-fucosylation activity of BfFucH on the deglycosylated proteins. Glyco-engineering is a strategy that can profoundly improve the safety and efficacy of biologics, including therapeutic monoclonal antibodies. Previously we identified a universal glycan that can be successfully conjugated to the GlcNAc-Asn297 site of the Fc domain of antibodies to enhance the ADCC, complement-dependent cytotoxicity (CDC), and anti-inflammatory activities.11 To further demonstrate that BfFucH can be applied to the preparation of other homogeneous antibodies, we used Humira as an example. The semi-quantification of glycopeptides from Humira was analyzed by MRM (Supplementary Table S1). The dominant glycoforms of commercial Humira are G0F and G1F accompanied with small amounts of Man5 and M3G0F (Figure 5b). After treatment with the cocktail of endoglycosidases and BfFucH simultaneously (Figure 5a), the glycan profile analysis showed that the heterogeneous mixture of N-glycans and the core fucose were efficiently hydrolyzed to give Humira with mono-GlcNAc residue in the Fc region (95% in Figure 5c). Without BfFucH, the majority of N-glycans remained fucosylated (89% in Figure 5c). Subsequently, the well-defined bi-antennary N-glycan oxazoline with two terminal sialic acids in the α-2,6-linkage was then ligated with the mono-GlcNAc residue of Humira (Figure 5d left and 5e) or the GlcNAc-Fuc residue of Humira (Figure 5d right and 5f) to obtain the respective homogeneous glycoforms in high yield (>95% N-SCT as shown in Figure 5e or >81% NF-SCT as shown in Figure 5f). These results show that BfFucH can efficiently remove the asparagine-linked core fucose from the Fc domain of IgG for further preparation of homogeneous antibody. BfFucH for N-glycan sequencing To understand how the glycan on a glycoprotein affects its structure and function, the sequences of the glycan at each glycosite has to be determined. Of many glycosidic linkages found in various glycoproteins, the fucosidic linkage is perhaps the most difficult to determine as it can be linked via the α-1,2, α-1,3/4, or α-1,6 linkage, giving various fucosylated N-glycans with great diversity. Different linkages and positions of fucoses may lead to different biological functions. The current biochemical method used to determine the precise position and linkage of fucose is based on different fucosidases: almond meal α-fucosidase (AMF) releases the α-1,3 and 8


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α-1,4-linked terminal fucose; Xanthomonas manihotis α-fucosidase (XMF) removes the α-1,2 linked fucoses; and bovine kidney α-fucosidase (BKF) releases the α-1,2 and α-1,6 linked fucoses at the non-reducing terminal and core region respectively.43-45 These fucosidases are widely used individually or in combination to determine the position and linkage of fucose in N-glycans, but still have their limitations. Our study showed that BfFucH has a high cleavage activity and selectivity toward different fucosidic bonds in the 2AB-tagged N-glycans. In order to further evaluate the BfFucH hydrolysis activity in glycopeptides and glycoproteins, we synthesized two glycopeptides, 0800F-P and 0823-P with fucose attached at different positions of the biantennary N-glycan for evaluation (Scheme 2a, 2b). The result showed that the fucose residue can be released only from the sample 0823-P with fucose linked to the outer GlcNAc residue, but not from the glycopeptide 0800F-P, where the fucose is bound to the innermost GlcNAc residue (Scheme 2c, 2d). This data suggest that the G0 structure of N-glycopeptide may shield and protect the core fucose from fucosidase hydrolysis as in the case of Rituximab and other glycoproteins. CONCLUSION Although numerous fucosidases have been studied with regard to their activities, none of them has been evaluated for the cleavage of the α-1,6-linked core fucose, except the two described by the Wang group. In this study, we reported a facile and practical method for the selective release of fucose from various glycans and glycoconjugates, and the synthesis of homogeneous glycoproteins without core fucose by utilizing the bacterial fucosidase BfFucH identified from a glycohydrolase library. We have carried out a comparative study of BfFucH and other fucosidases in combination with an endo-glycosidase or a cocktail and conclude that the recombinant BfFucH is the most efficient and cost-effective enzyme for the cleavage of the α-1,6-linked core fucose to convert heterogeneous glycoproteins to homogeneous mono-GlcNAc glycoform, including antibody, Fc fusion protein, erythropoietin, interferon, and the influenza virus surface glycoprotein hemagglutinin (HA). In addition, the mono-GlcNAc antibodies have been further used as substrates for transglycosylation to prepare homogenous antibodies with well-defined glycan,6, 9, 46, 47 and a mono-GlcNAc hemagglutinin has been used for the development of universal influenza vaccine.48 We have also shown that BfFucH is potentially useful for glycan sequencing and analysis of more complex glycoconjugates.

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METHODS Materials All chemical and reagents were purchased from Sigma-Aldrich and Merck except the following: anti-tumor necrosis factor-alpha (TNFα) antibody and Adalimumab (Humira) were purchased from AbbVie; anti-human CD20 mouse/human chimeric IgG1 Rituximab (Rituxan) was purchased from Genentech and IDEC Pharmaceutical; the TNF receptor-Fc fusion protein Etanercept (Enbrel) was purchased from Wyeth Pharmaceuticals; Epoetin beta (Recormon) was purchased from Hoffmann-La Roche; Interferon β1a (Rebif) was purchased from EMD Serono; para-Nitrophenyl α- or β-glycoside, Lewis sugars, blood type sugars and human milk oligosaccharides were purchased from Carbosynth; biotinylated Aleuria Aurantia Lectin (AAL) and HRP-Conjugated Streptavidin were purchased from Vector Laboratory. All bacterial genomic DNAs were purchased from American Type Culture Collection (ATCC). α-L-fucosidaes from bovine kidney (F-5884, Lot: SLBL8171V, 12.5 units/mg) was purchased from Sigma-Aldrich. Chemiluminescence on protein blots was visualized and quantified using the ImageQuant LAS 4000 biomolecular imager system. Evaluation of the substrate specificity of BfFucH by UPLC Each 2-AB labeled glycan (10 pmol) was mixed with BfFucH (1 µg) in a 10 µL reaction solution (50 mM sodium phosphate buffer, pH 7.0), and the mixture was incubated at 37 °C overnight. Glycans were then subjected to UPLC separation [ACQUITY UPLC H-class System and ACQUITY UPLC BEH Glycan column (130 Å, 1.7 µm, 2.1 x 150 mm) from Waters] and fluorescence detection (Ex/Em: 330 nm/420 nm) after cleanup with C4 zip-tips. The percentage of fucose removal in each reaction was measured and calculated based on the shift of retention time and the relative intensity of 2-AB detected from the glycan and its defucosylated product. The activity of BfFucH and that of commercial bovine α-L-fucosidase were compared 30 ug of Rituxan and 3 ug of endoS (wild type) were incubated in a 50 mM sodium phosphate buffer, pH 7.0, in 37 0C for 2 h prior to addition of 30 mU of BfFucH or bovine α-L-fucosidase. Samples were taken in 2, 4, and 8 h for AAL lectin blotting, and the sample band was quantified by Bio-RAD Image Lab version 5.2 build 14.

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Generation of mono-GlcNAc or GlcNAc-(Fuc α-1,6) of immunoglobulin G, Fc-fusion protein, EPO, interferon (IFNβ1a), influenza hemagglutinin (HA) and Humira All glycoproteins underwent buffer exchange with 50 mM sodium phosphate buffer (pH 7.0). The cocktail endoglycosidase solution, including F1, F2, F3, H and S (1mg/mL), was added first in order to remove all the N-glycan chains except the GlcNAc bound to the Asn residue of glycoproteins, followed by a suitable quantity of fucosidase. The solution was incubated at 37 °C for 48 h to completely remove the core fucose bound to the GlcNAc residue of glycoproteins. The fucose content was determined by trypsin digestion of deglycosylated glycoprotein followed by LC-MS analysis.

SUPPORTING INFORMATION Supporting Information Available: This material is available free of charge via the Internet.

ACKNOWLEDGMENTS We thank C.-Y. Wu for the study of hemagglutinin and M.-I. Lin for the preparation of MRM data. T.-I. Tsai thanks Taiwan Bio-Development Foundation for a partial scholarship. This work was supported by the Academia Sinica, Taiwan and National Institute of Health grant AI072155 to CHW.

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4. Durocher, Y., and Butler, M. (2009) Expression systems for therapeutic glycoprotein production, Curr. Opin. Biotechnol. 20, 700-707. 5. Anthony, R. M., Nimmerjahn, F., Ashline, D. J., Reinhold, V. N., Paulson, J. C., and Ravetch, J. V. (2008) Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc, Science 320, 373-376. 6. Zou, G., Ochiai, H., Huang, W., Yang, Q., Li, C., and Wang, L.-X. (2011) Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor, J. Am. Chem. Soc. 133, 18975-18991. 7. DiLillo, David J., and Ravetch, Jeffrey V. (2015) Differential Fc-receptor engagement drives an anti-tumor vaccinal effect, Cell 161, 1035-1045. 8. Shields, R. L., Lai, J., Keck, R., O'Connell, L. Y., Hong, K., Meng, Y. G., Weikert, S. H. A., and Presta, L. G. (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity, J. Biol. Chem. 277, 26733-26740. 9. Huang, W., Giddens, J., Fan, S.-Q., Toonstra, C., and Wang, L.-X. (2012) Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions, J. Am. Chem. Soc. 134, 12308-12318. 10. Goodfellow, J. J., Baruah, K., Yamamoto, K., Bonomelli, C., Krishna, B., Harvey, D. J., Crispin, M., Scanlan, C. N., and Davis, B. G. (2012) An endoglycosidase with alternative glycan specificity allows broadened glycoprotein remodelling, J. Am. Chem. Soc. 134, 8030-8033. 11. Lin, C.-W., Tsai, M.-H., Li, S.-T., Tsai, T.-I., Chu, K.-C., Liu, Y.-C., Lai, M.-Y., Wu, C.-Y., Tseng, Y.-C., Shivatare, S. S., Wang, C.-H., Chao, P., Wang, S.-Y., Shih, H.-W., Zeng, Y.-F., You, T.-H., Liao, J.-Y., Tu, Y.-C., Lin, Y.-S., Chuang, H.-Y., Chen, C.-L., Tsai, C.-S., Huang, C.-C., Lin, N.-H., Ma, C., Wu, C.-Y., and Wong, C.-H. (2015) A common glycan structure on immunoglobulin g for enhancement of effector functions, Proc. Natl. Acad. Sci. U.S.A. 112, 10611-10616. 12. Ferrara, C., Grau, S., Jäger, C., Sondermann, P., Brünker, P., Waldhauer, I., Hennig, M., Ruf, A., Rufer, A. C., Stihle, M., Umaña, P., and Benz, J. (2011) Unique carbohydrate–carbohydrate interactions are requirsed for high affinity binding between FcγRIII and antibodies lacking core fucose, Proc. Natl. Acad. Sci. U.S.A. 108, 12669-12674. 13. Ferrara, C., Brünker, P., Suter, T., Moser, S., Püntener, U., and Umaña, P. (2006) Modulation of therapeutic antibody effector functions by glycosylation engineering: Influence of Golgi enzyme localization domain and co-expression 12


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modulation of antibody fucosylation with small molecule fucostatin inhibitors and cocrystal structure with GDP-mannose 4,6-dehydratase, ACS Chemical Biology 11, 2734-2743. 23. Shivatare, S. S., Chang, S.-H., Tsai, T.-I., Tseng, S. Y., Shivatare, V. S., Lin, Y.-S., Cheng, Y.-Y., Ren, C.-T., Lee, C.-C. D., Pawar, S., Tsai, C.-S., Shih, H.-W., Zeng, Y.-F., Liang, C.-H., Kwong, P. D., Burton, D. R., Wu, C.-Y., and Wong, C.-H. (2016) Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of hiv antibodies, Nat Chem 8, 338-346. 24. Ashida, H., Miyake, A., Kiyohara, M., Wada, J., Yoshida, E., Kumagai, H., Katayama, T., and Yamamoto, K. (2009) Two distinct α-L-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates, Glycobiology 19, 1010-1017. 25. Katayama, T., Sakuma, A., Kimura, T., Makimura, Y., Hiratake, J., Sakata, K., Yamanoi, T., Kumagai, H., and Yamamoto, K. (2004) Molecular cloning and characterization of Bifidobacterium bifidum 1,2-α-L-fucosidase (afca), a novel inverting glycosidase (glycoside hydrolase family 95), J. Bacteriol. 186, 4885-4893. 26. Wong-Madden, S. T., and Landry, D. (1995) Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas, Glycobiology 5, 19-28. 27. Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., and Henrissat, B. (2009) The carbohydrate-active enzymes database (CAZY): An expert resource for glycogenomics, Nucleic Acids Res. 37, D233-D238. 28. Danishefsky, S. J., Shue, Y.-K., Chang, M. N., and Wong, C.-H. (2015) Development of Globo-H cancer vaccine, Acc. Chem. Res. 48, 643-652. 29. Lee, H.-Y., Chen, C.-Y., Tsai, T.-I., Li, S.-T., Lin, K.-H., Cheng, Y.-Y., Ren, C.-T., Cheng, T.-J. R., Wu, C.-Y., and Wong, C.-H. (2014) Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen, J. Am. Chem. Soc. 136, 16844-16853. 30. Sulzenbacher, G., Bignon, C., Nishimura, T., Tarling, C. A., Withers, S. G., Henrissat, B., and Bourne, Y. (2004) Crystal structure of Thermotoga maritima α-L-fucosidase, J. Biol. Chem. 279, 13119-13128. 31. Stocker, B. L., Jongkees, S. A. K., Win-Mason, A. L., Dangerfield, E. M., Withers, S. G., and Timmer, M. S. M. (2013) The ‘mirror-image’ postulate as a guide to the selection and evaluation of pyrrolidines as α-L-fucosidase inhibitors, Carbohydr. Res. 367, 29-32. 32. Sakurama, H., Tsutsumi, E., Ashida, H., Katayama, T., Yamamoto, K., and Kumagai, H. (2012) Differences in the substrate specificities and active-site 14


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structures of two α-L-fucosidases (glycoside hydrolase family 29) from Bacteroides thetaiotaomicron, Bioscience, Biotechnology, and Biochemistry 76, 1022-1024. 33. Sakurama, H., Fushinobu, S., Hidaka, M., Yoshida, E., Honda, Y., Ashida, H., Kitaoka, M., Kumagai, H., Yamamoto, K., and Katayama, T. (2012) 1,3-1,4-α-L-fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains, J. Biol. Chem. 287, 16709-16719. 34. Gregg, K. J., Finn, R., Abbott, D. W., and Boraston, A. B. (2008) Divergent modes of glycan recognition by a new family of carbohydrate-binding modules, J. Biol. Chem. 283, 12604-12613. 35. Wright, D. W., Moreno-Vargas, A. J., Carmona, A. T., Robina, I., and Davies, G. J. (2013) Three dimensional structure of a bacterial α-L-fucosidase with a 5-membered iminocyclitol inhibitor, Biorg. Med. Chem. 21, 4751-4754. 36. Hottin, A., Wright, D. W., Steenackers, A., Delannoy, P., Dubar, F., Biot, C., Davies, G. J., and Behr, J.-B. (2013) α-L-fucosidase inhibition by pyrrolidine– ferrocene hybrids: Rationalization of ligand-binding properties by structural studies, Chem. Eur. J. 19, 9526-9533. 37. Léonard, R., Pabst, M., Bondili, J. S., Chambat, G., Veit, C., Strasser, R., and Altmann, F. (2008) Identification of an arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides, Phytochemistry 69, 1983-1988. 38. Lammerts van Bueren, A., Ardèvol, A., Fayers-Kerr, J., Luo, B., Zhang, Y., Sollogoub, M., Blériot, Y., Rovira, C., and Davies, G. J. (2010) Analysis of the reaction coordinate of α-L-fucosidases: A combined structural and quantum mechanical approach, J. Am. Chem. Soc. 132, 1804-1806. 39. Collin, M., and Olsen, A. (2001) EndoS, a novel secreted protein from streptococcus pyogenes with endoglycosidase activity on human IgG, EMBO J. 20, 3046-3055. 40. Li, T., Tong, X., Yang, Q., Giddens, J. P., and Wang, L.-X. (2016) Glycosynthase mutants of endoglycosidase s2 show potent transglycosylation activity and remarkably relaxed substrate specificity for antibody glycosylation remodeling, J. Biol. Chem. 291, 16508-16518. 41. Wang, X., Inoue, S., Gu, J., Miyoshi, E., Noda, K., Li, W., Mizuno-Horikawa, Y., Nakano, M., Asahi, M., Takahashi, M., Uozumi, N., Ihara, S., Lee, S. H., Ikeda, Y., Yamaguchi, Y., Aze, Y., Tomiyama, Y., Fujii, J., Suzuki, K., Kondo, A., Shapiro, S. D., Lopez-Otin, C., Kuwaki, T., Okabe, M., Honke, K., and Taniguchi, N. (2005) Dysregulation of TGF-β1 receptor activation leads to 15



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abnormal lung development and emphysema-like phenotype in core fucose-deficient mice, Proc. Natl. Acad. Sci. U.S.A. 102, 15791-15796. 42. Chen, C.-Y., Jan, Y.-H., Juan, Y.-H., Yang, C.-J., Huang, M.-S., Yu, C.-J., Yang, P.-C., Hsiao, M., Hsu, T.-L., and Wong, C.-H. (2013) Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer, Proc. Natl. Acad. Sci. U.S.A. 110, 630-635. 43. Marino, K., Bones, J., Kattla, J. J., and Rudd, P. M. (2010) A systematic approach to protein glycosylation analysis: A path through the maze, Nat Chem Biol 6, 713-723. 44. Saldova, R., Piccard, H., Pérez-Garay, M., Harvey, D. J., Struwe, W. B., Galligan, M. C., Berghmans, N., Madden, S. F., Peracaula, R., Opdenakker, G., and Rudd, P. M. (2013) Increase in sialylation and branching in the mouse serum N-glycome correlates with inflammation and ovarian tumour progression, PLoS ONE 8, e71159. 45. Tharmalingam, T., Adamczyk, B., Doherty, M., Royle, L., and Rudd, P. (2013) Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides, Glycoconjugate J. 30, 137-146. 46. Wang, L.-X., and Davis, B. G. (2013) Realizing the promise of chemical 47.

glycobiology, Chemical Science 4, 3381-3394. Wang, L.-X. (2011) The amazing transglycosylation endo-beta-n-acetylglucosaminidases, Glycotechnology 23, 33-52.

Trends

in

activity

Glycoscience

of and

48. Chen, J. R., Yu, Y. H., Tseng, Y. C., Chiang, W. L., Chiang, M. F., Ko, Y. A., Chiu, Y. K., Ma, H. H., Wu, C. Y., Jan, J. T., Lin, K. I., Ma, C., and Wong, C. H. (2014) Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections, Proc Natl Acad Sci U S A 111, 2476-2481.

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TABLE, SCHEMES, AND FIGURES

Table 1. Substrate specificity of BfFucH evaluated by UPLC Each 2-AB labeled glycan (10 pmol) was mixed with BfFucH (1 µg) in a 10 µL reaction solution (50 mM sodium phosphate buffer, pH 7.0), and the mixture was incubated at 37 °C overnight. Glycans were then subjected to UPLC separation [ACQUITY UPLC H-class System and ACQUITY UPLC BEH Glycan column (130 Å, 1.7 m, 2.1 x 150 mm) from Waters] and fluorescence detection (Ex/Em: 330 nm/420 nm) after cleanup with C4 zip-tips. The percentage of fucose removal in each reaction was measured and calculated based on the shift of retention time and the relative intensity of 2-AB detected from the glycan and its defucosylated product. Symbol

Sugar name Galactose (Gal) N-acetylgalactosamine (GalNAc) N-acetylglucosamine (GlcNAc) Mannose (Man) Fucose (Fuc) N-acetylneuraminic acid (Neu5Ac)

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Glycan Name

A2F

NA2F

NGA2F

M3N2F

M1N2F

26.3±6.4

21.5±2.1

25.8±2.4

35.0±2.2

53.3±6.0

LNFP I

LNFP II

LNFP III

Lewis a

Lewis b

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0823

133

143

146

Complete

Complete

Complete

Complete

digestion

digestion

digestion

digestion

Lewis x

Lewis y

Glycan Structure

% Fucose Removal Glycan Name

Sialyl Globo H Lewis x

Glycan Structure

Complete

% Fucose 80.0±3.6

2.3±2.1

3.7±2.9

No digestion

7.7±1.2

No digestion

Removal

digestion (α1,2-Fuc)

No digestion

Complete digestion

18


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Scheme 1. α-L-Fucose-containing glycoconjugates. Fucose is present in many glycoconjugates with α-1,2/3/4/6 linkages in various glycans

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Scheme 2. Chemoenzymatic synthesis of glycopeptides for use in glycan sequencing. (a) Upper panel: chemoenzymatic synthesis of glycopeptide 0800F-P by reaction of the glycan CT-oxazoline with the GlcNAc-containing glycopeptide catalyzed by an endoM mutant (N175Q) followed by galactosidase and fucosyltransferase 8 (Fut8) catalyzed fucosylation. Lower panel: MS spectrum of glycopeptide 0800F-P. (b) Upper panel: chemoenzymatic synthesis of glycopeptide 0823-P by reaction of the oxazoline form of N-glycan containing α-1,3-fucose (attached to the α6 arm GlcNAc) with the GlcNAc-containing glycopeptide catalyzed by endoM N175Q. Lower panel: MS spectrum of glycopeptide 0823-P. (c) Evaluation of the substrate specificity of BfFucH using two different fucose-containing glycopeptides. BfFucH can only hydrolyze the outer fucose in glycopeptide 0823-P, but not the core fucose on glycopeptide 0800F-P. (d) MS spectrum of glycopeptide 0823-P after defucosylation by BfFucH. Upper panel: defucosylation product of glycopeptide 0823-P by BfFucH. Lower panel: glycopeptide 0823-P without BfFucH treatment. (a)

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(b)

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(c)

(d)

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Figure 1. Biochemical properties and specificity of BfFucH (a) pH profile of BfFucH (b) Metal ion influence on BfFucH (c) Profile of BfFucH at different temperatures (d) Kinetic study of BfFucH: kcat = 183.8 s-1, Km = 437.0 uM, kcat/Km = 0.42 s-1 uM-1, Vmax = 11.03 mM min-1 mg-1. The substrate used in this study is p-Nitrophenyl α-L-fucopyranoside (pNP-α-L-Fuc).

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Figure 2. Comparison of the defucosylaton activity between BfFucH and various reported fucosidases. The defucosylation activities of BfFucH and various reported fucosidases toward endoS-treated Rituxan were evaluated and compared. Mixtures of Rituxan and endoS were incubated with each fucosidase, then samples were taken at different times to evaluate the defucosylation activsity by AAL lectin blotting. The fucosidase can not remove the core fucose without endo-glycosidase treatment and this is used as negative control. For details see the Material and Methods session.

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Figure 3. Time course of BfFucH-catalyzed defucosylation of Rituxan. The deglycosylated Rituxan was used for the defucosylation activity evaluation of BfFucH. Mixtures of BfFucH and deglycosylated Rituxan were incubated at different temperatures and aliquots were taken at different times to evaluate the defucosylation activity by AAL lectin blotting. The staining intensity is highly relevant to the content of core fucose. For details see the Material and Methods session.

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Figure 4. Hydrolysis activity of BfFucH using various glycoforms of glycoproteins. SDS-PAGE of glycoproteins: Humira (Antibody, AbbVie), Rituxan (Antibody, Roche), Enbrel (Fc-fusion protein, Pfizer), Erythropoietin (EPO, Roche), Rebif (Interferon beta-1a, EMD Serono), Influenza A H1N1 (A/WSN/33) hemagglutinin (Recombinant protein, expressed from HEK293T). (b) AAL lectin blotting of various glycoproteins. (c) Western blotting of glycoproteins by specific primary antibody. Lane 1: sample protein without any treatment as control; Lane 2: sample protein treated with a cocktail of endoglycosidases; Lane 3: sample protein treated with a cocktail of endoglycosidases and BfFucH; Lane 4: sample protein treated with PNGaseF as negative control. The core fucose was released by treatment with a cocktail of endoglycosidases and BfFucH as described in the experiment. HC: antibody heavy chain; LC: antibody light chain.

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Figure 5. Glyco-engineering of de-fucosylated homogeneous antibody Humira. (a) SDS-PAGE of intact, endoglycosidase cocktail treatment, and endoglycosidase cocktail coupled with BfFucH treatment of Humira (Lane 1, 2, and 3, respectively). (b) Glycan profile of intact Humira. (c) Glycan profiles of Humira after endoglycosidase cocktail coupled with BfFucH treatment and endoglycosidase cocktail treatment only. (d) Glycoengineering of de-fucosylated Humira (Hu-N) and fucosylated Humira (Hu-NF). Lane 4 is Humira treated with the endoglycosidase cocktail and BfFucH, while Lane 5 is the transglycosylation product prepared from the de-fucosylated mono-GlcNAc-Humira and SCT-oxazoline by endoS D233Q. Lane 6 is Humira treated with the endoglycosidase cocktail, while Lane 7 is the transglycosylation product prepared from de-glycosylated Humira and SCT-oxazoline by endoS D233Q. (e) Glycan profile of the transglycosylation product with BfFucH treatment (Lane 5). (f) Glycan profile of the transglycosylation product without BfFucH treatment (Lane 7). N means mono-GlcNAc; NF means GlcNAc with fucose (α-1,6 linkage); N-SCT means α-2,6-disialylated-galacto-biantennary complex N-glycan; NF-SCT means α-2,6-disialylated-galacto-biantennary complex N-glycan with core fucose.

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Graphical Table of Contents

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Graphical Table of Contents 338x190mm (96 x 96 DPI)


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An Effective Bacterial Fucosidase for Glycoprotein Remodeling - ACS

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