Application of Human Glycosyltransferases in N-Glycan Synthesis and Their Substrate Specificity Studies

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Chemistry Dissertations Department of Chemistry

12-15-2016

Application of Human Glycosyltransferases in N-glycan Synthesis and Their Substrate Specificity Studies

Angie Dayan Calderon Molina Georgia State University

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Recommended Citation Calderon Molina, Angie Dayan, "Application of Human Glycosyltransferases in N-glycan Synthesis and Their Substrate Specificity Studies." Dissertation, Georgia State University, 2016. https://scholarworks.gsu.edu/chemistry_diss/128

This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. APPLICATION OF HUMAN GLYCOSYLTRANSFERASES IN N-GLYCAN SYNTHESIS

AND THEIR SUBSTRATE SPECIFICITY STUDIES

ANGIE DAYAN CALDERON MOLINA

Under the Direction of Peng G. Wang, PhD

ABSTRACT

Glycoscience is important in many areas such as human health, energy and material science.

Glycans have been shown to be involved in the pathophysiology of almost every major disease.

Additional glycan structure knowledge is required to help advance personal medicine, and pharmaceutical developments, among others. For glycoscience to advance there is a need for large quantities of well-defined glycans and have quick access to glycosyltransferases for manipulating glycan synthesis.

Herein, we will cover our efforts on studying the substrate specificities of human glycosyltransferases such as FUT8 and Gn-T V, and their application on N-glycan synthesis.

Complex asymmetric N-glycan isomer structures have been related to many diseases such as breast cancer, among others. Synthesis of complex asymmetric N-glycan isomer structures including: a1,6 core-fucosylated, and tri-antennary structures can be achieved by taking advantage of the high specificity of glycosyltransferases that can work as unique catalyst to generate well-defined glycan structures.

INDEX WORDS: FUT8, fucosylation, N-glycan, glycosyltransferases, Gn-T I, Gn-T II, Gn-T V. APPLICATION OF HUMAN GLYCOSYLTRANSFERASES IN N-GLYCAN SYNTHESIS

AND THEIR SUBSTRATE SPECIFICITY STUDIES

ANGIE DAYAN CALDERON MOLINA

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

in the College of Arts and Sciences

Georgia State University

2016

AND THEIR SUBSTRATE SPECIFICITY STUDIES

ANGIE DAYAN CALDERON MOLINA

Committee Chair: Peng George Wang

Committee: Binghe Wang

Jenny Yang

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

December 2016 iv

DEDICATION

I would like to dedicate this work to my family and friends who supported me throughout my time in graduate school. I would especially like to thank my parents, Fernando Calderon and

Beatriz Molina for their unconditional support, encouragement and love. I would also like to thank Xuan Wang for his patience, love, and encouragement during all of this time. Finally, I want to dedicate this dissertation to my grandparents who would have been very proud of this accomplishment Jesus Molina and Aura Rodriguez. v

ACKNOWLEDGEMENTS

First and foremost, I would like to sincerely thank my advisor Dr. Peng George Wang. I am especially grateful for his guidance, encouragement and support throughout all these years of hard work and struggles. I would also like to thank Dr. Binghe Wang, who has also been supportive every step of the way and has seen me grow and mature since my undergraduate years in his research lab. I would also like to thank Dr. Jenny Yang, for all her support and knowledge. I want to thank Dr. Barbara Baumstark and Dr. Al Baumstark for all of their encouragement, understanding, support all throughout my academic career at GSU. I want to especially thank my collaborator Dr. Lei Li for all his guidance and knowledge, without his support this work could not have been done. Thanks to all of the Wang group lab members especially Yunpeng, Jun, Cheng, Jingyao, Wanyi, Zhigang, Yuxi, Xu, Hailiang, Xuan, Kristina,

Shanshan, and all others for their help in my research. Last but not least I want to thank the Bio-

Bus program, coordinators, all the fellows and volunteers who have encourage me and helped me become a better teacher.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... 5

LIST OF FIGURES ...... 6

1 INTRODUCTION ...... 11

1.1 Purpose ...... 11

1.2 Introduction ...... 12

1.3 Glycosylation importance ...... 14

1.4 N-Glycosylation site ...... 14

1.5 N-Glycosylation difference ...... 15

1.6 Protein glycosylation pathway ...... 16

1.7 Therapeutic N-linked glycoproteins ...... 18

1.8 References: ...... 19

2 SUBSTRATE SPECIFICITY OF FUT8 AND CHEMOENZYMATIC

SYNTHESIS OF CORE-FUCOSYLATED ASYMMETRIC N-GLYCANS ...... 21

2.1 Abstract ...... 21

2.2 Introduction ...... 21

2.3 Results and Discussion ...... 24

2.4 Conclusion ...... 30

2.5 Acknowledgements ...... 30

2.6 Experimental ...... 31

2.6.1 Materials and enzymes ...... 31

2.6.2 Expression and purification of human FUT8 ...... 31

2.6.3 Substrate specificity study of FUT8 ...... 33

2.6.4 General methods for glycan synthesis and purification ...... 36

2.6.5 HPLC profiles and MS data of purified N-glycans ...... 37

2.6.6 ESI-MS and 1H NMR data of purified N604 ...... 53

2.6.7 NMR spectra and data of purified core-fucosylated N-glycans ...... 54

2.7 References ...... 60

3 FUT8: FROM BIOCHEMISTRY TO SYNTHESIS OF CORE-

FUCOSYLATED N-GLYCANS...... 62

3.1 Abstract ...... 62

3.2 Introduction ...... 63

3.3 FUT8 specificity studies ...... 65

3.4 Traditional versus chemo-enzymatic synthesis of core fucosylated N-

glycans. 67

3.5 Conclusion ...... 74

3.6 Acknowledgements ...... 75

3.7 References ...... 75

4 ENZYMATIC SEPARATION OF ASYMMETRIC BI- AND TRI-

ANTENNARY N-GLYCAN ISOMERS USING FUT8 AND GN-TV ...... 78

4.1 Abstract ...... 78

4.2 Introduction ...... 78

4.3 Results and Discussion ...... 81

4.3.1 Generation of asymmetric biantennary and triantennary N-glycans ...... 82

4.3.2 Substrate Specificity for human Gn-T V ...... 83

4.3.3 Enzymatic separation of N-glycan isomers ...... 84

4.4 Conclusion ...... 89

4.5 Acknowledgements ...... 89

4.6 Experimental ...... 89

4.6.1 Materials and enzymes ...... 89

4.6.2 Expression and Purification ...... 90

4.6.3 Generation of starting materials N001 and N3001 from natural source ... 92

4.6.4 Generation of asymmetric structures using LacZ ...... 93

4.6.5 Separation of N-glycan isomers using FUT8 and Gn-T V ...... 95

4.6.6 N-glycan labeling using 2-AA and AEAB ...... 97

4.6.7 Substrate specificity studies on b-galactosidase (LacZ) ...... 98

4.6.8 Substrate specificity studies on Gn-T V ...... 99

4.6.9 Substrate specificity studies of Gn-T V on triantennary N-glycans ...... 101

4.6.10 General methods for asymmetric glycan synthesis from natural products

and purification ...... 101

4.6.11 HPLC profiles and MS data of purified N-glycans ...... 103

4.6.12 NMR spectra and data of purified asymmetric biantennary and

triantennary N-glycans ...... 112

4.7 References ...... 119

5 CONCLUSIONS ...... 123

APPENDICES ...... 124

Appendix A Additional human glycosyltransferases expressed in Sf9 using TIPs method. (Gn-T I, Gn-T II, and Gn-T III) ...... 124

Appendix A.1 SDS-PAGE for Gn-T I and Gn-T II ...... 124

Appendix A.2 Western-Blot for Gn-T III ...... 124

Appendix A.3 Activity Assay for Gn-T I, Gn-T II and Gn-T III ...... 125

Appendix B Additional chemo-enzymatic synthesized a1,6-core-fucosylated N- glycan structures ...... 127

Appendix B.1 Scheme for chemo-enzymatic synthesis of additional complex

symmetric core-fucosylated N-glycans...... 128

Appendix B.2 HPLC profiles and MS data of purified complex symmetric N-

glycans ...... 128

LIST OF TABLES

Table 2.6.3.1 Substrate specificity of FUT8. ND, Not Detected. Conversions were monitored by

HPLC-ELSD, average of three replicates were shown...... 34

Table 2.6.7.1 % identity of FUT8 between animal species.26, 27 ...... 64

Table 4.6.7.1 Substrate specificity of LacZ. ND, Not Detected. Conversions were monitored by

HPLC-RF, average of three replicates were shown...... 99

Table 4.6.8.1 Summary of specificity studies for Gn-T V. All of the substrates were AEAB

labeled. * complete conversion after prolonged incubation...... 100

Table 4.6.9.1 Gn-T V substrate specificity on triantennary N-glycans. ND, Not Detected.

Conversions were monitored by HPLC-RF, average of three replicates were shown. ... 101

LIST OF FIGURES

Figure 1.2.1 Glycosylation types...... 13

Figure 1.2.2 N-glycan structures found in mature glycoprotein...... 13

Figure 1.6.1 Summary of processing and maturation of an N-glycan protein from the ribosome

through the ER and Golgi apparatus to its final destination.8 ...... 17

Figure 2.2.1 FUT8 catalyzed reaction and substrate specificity of human FUT8 using 77 N-

glycans as potential acceptors (see Figure 2.6.3 for the full list of N-glycans, Table

2.6.3.1 for conversion percentage towards each glycan). The average conversion

percentage of three replicates for each acceptor was shown...... 24

Figure 2.3.1 Two N-glycans (m/z) identified from CA125 and possible N-glycans (black

squared). Red dash squared N-glycan structures were synthesized in this study...... 26

Figure 2.3.2 Chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans from N110

(A), and MALDI-MS analysis of purified glycans (B). Reaction conditions: a) FUT8,

GDP-Fuc, FastAP; b) b4GALT1, UDP-Gal, FastAP, Mn2+; c) 30% ammonium hydroxide

: H2O (1 : 10), 6 h; d) PmST1m, CMP-Neu5Ac; e) Pd2,6ST, CMP-Neu5Ac; f)

Hpa1,3FT, GDP-Fuc, FastAP, Mn2+. FUT8, human a1,6-fucosyltransferase; b4GALT1,

bovine b1,4-galactosyltransferase; PmST1m, Pasteurella multocida a2,3-

sialyltransferase 1 mutant E271F/R313Y; Pd2,6ST, Photobacterium damselae a 2,6-

sialyltransferase; Hpa1,3FT, c-terminal 66 amino acids truncated Helicobacter pylori

a1,3-fucosyltransferase; FastAP, thermo-sensitive alkaline phosphatase...... 28

Figure 2.3.3 Enzymatic synthesis of core-fucosylated N-glycans starting with N210. (See Figure

2.3.2A) for detailed description of each step...... 29

Figure 2.6.1 SDS-PAGE and western blot analysis of FUT8. 1) SDS-PAGE of medium after

overexpression of FUT8; 2) SDS-PAGE of purified FUT8; and 3) western blot analysis

of purified FUT8 using anti-His antibody as the primary antibody. BSA, a major protein

component of Fetal Bovine Serum used in the culture medium, has similar molecular

weight as that of FUT8...... 33

Figure 2.6.2 MALDI-MS analysis of FUT8 catalyzed reaction using N000 as substrate. Majority

of N000 (molecular weight 1316.4865, found M+Na 1339.4568) was converted into core-

fucosylated one (molecular weight 1462.5444, found M+Na 1485.5385)...... 33

Figure 2.6.3 The 77 N-glycans used as acceptor for FUT8 substrate specificity study. The

preparation and characterization of all substrates were published previously6. The ones in

the red square were found to be suitable acceptors for FUT8...... 35

Figure 3.2.1 Sample reaction catalyzed by a1,6 fucosyltransferase (FUT8)...... 63

Figure 3.4.1 Chemo-enzymatic synthesis of core-fucosylated glycopeptide and glycoprotein

using Endo-F3 and mutated Endo-F3 D165A to transfer biantennary and triantennary

complex N-glycans.66, 67 ...... 69

Figure 3.4.2 A) CeFUT8 specificity assay using structures on glycan array. B) All substrates

were chemically synthesized with linker. C&D) Compound 5 and 7 were synthesized

using CeFUT8 from A9 and A14 respectively, in solution and the structure was

confirmed by NMR and MS.47 ...... 70

Figure 3.4.3 Scheme of chemo-enzymatic synthesis of 14 a1,6 core fucosylated invertebrate and

plant N-glycans containing xylose.50 Reaction Conditions: a. CeFUT8, b. GalT, c.

AtFucTA/CeFUT1, d. GalNAcT, e. CeFUT6, f. N-acetyl-glucosaminidase...... 71

Figure 3.4.4 Compound 2 before deprotection of R1=Bn by hydrogenation. In red, incorporated

13C isotopes. Scheme of chemo-enzymatic synthesis of CF-glycans.6, 49 Reaction

Conditions: a. b-1,4-GalT, b. 10% Pd/C, H2 c. a1,6 FucT d. N-acetyl-glucosaminidase, e.

a-2,3-SialylT...... 72

Figure 3.4.5 CF-glycans generated by Wang lab.48 ...... 73

Figure 3.4.6 (A) Scheme for chemo-enzymatic synthesis of asymmetric a1,6 core-fucosylated N-

glycans starting with N110 as a substrate. B) Scheme for chemo-enzymatic synthesis of

asymmetric a1,6 core fucosylated N-glycans starting with N210 as a substrate.45

Reaction conditions: a. FUT8, GDP-Fuc, FastAP; b. b4GALT1, UDP-Gal, FastAP, Mn2+;

c. 30% ammonium hydroxide : H2O (1 : 10), 6 h; d) PmST1m, CMP-Neu5Ac; e.

Pd2,6ST, CMP-Neu5Ac; f. Hpa1,3FT, GDP-Fuc, FastAP, Mn2+. FUT8, human a1,6-

fucosyltransferase; b4GALT1, bovine b1,4-galactosyltransferase; PmST1m, Pasteurella

multocida a2,3-sialyltransferase 1 mutant E271F/R313Y; Pd2,6ST, Photobacterium

damselae a 2,6-sialyltransferase; Hpa1,3FT, c-terminal 66 amino acids truncated

Helicobacter pylori a1,3-fucosyltransferase; FastAP, thermo-sensitive alkaline

phosphatase...... 74

Figure 4.2.1 Gn-T V catalyzed reaction and substrate specificity of human Gn-T V using 17 N-

glycans as potential acceptors,(See Table 4.6.8.1 and Table 4.6.9.1 for conversion

percentage towards each glycan). The average conversion percentage of three replicates

for each acceptor was shown...... 81

Figure 4.3.1 A. Scheme for the synthesis of possible asymmetric N-glycan isomers from SGP

from isolated from egg yolk. B. Scheme for the synthesis of possible asymmetric N-

glycan isomers from fetal bovine serum. N-glycan boxed in red are the identified

asymmetric N-glycan generated from lacZ digestion...... 82

Figure 4.3.2 A. LacZ 3 h digestion of N001 and identification of possible isomer intermediate

using FUT8 and Gn-T V. Reactions were monitored by MALDI-MS. B. LacZ 3 h

digestion intermediate incubated with FUT8 overnight. C. LacZ 3 h digestion

intermediate incubated with Gn-T V overnight. D. Positive control N211 substrate

incubated with FUT8 overnight. D. Negative control N111 substrate incubated with

FUT8 overnight...... 85

Figure 4.3.3 Scheme of possible isomer structures without 1 and 2 galactose residues after LacZ

digestion...... 86

Figure 4.3.4 A. Scheme for identification of possible isomer intermediate using FUT8 and Gn-T

V. B. MALDI-MS analysis of N3001 without 1 galactose residue after overnight

incubation with Gn-T V. No a1,6 core-fucosylated product detected after overnight

incuation with FUT8. Reaction conditions: a) FUT8, GDP-Fuc, FastAP; b) Gn-T V,

UDP-GlcNAc, Mg2+; BSA, Tritron 100-X. Red box indicates the isomers identified from

the MALDI-MS results (See pg.93 §4.6.11)...... 87

Figure 4.3.5 A. Scheme for identification of possible isomer intermediate using FUT8 and Gn-T

V. B. MALDI-MS analysis of N3001 without 2 galactose residue after overnight

incubation with FUT8 (N36361) and Gn-T V (N37111-AEAB). Reaction conditions: a)

FUT8, GDP-Fuc, FastAP; b) Gn-T V, UDP-GlcNAc, Mg2+; BSA, Tritron 100-X. Red

box indicates the isomers identified from the MALDI-MS results (See pg.93 §4.6.11). . 88

Figure 4.6.1 Western Blot for Gn-T V...... 91

Figure 4.6.2 SDS-PAGE for Ec-LacZ expression and purification M. Premixed Protein Marker

(High) (44.3-200 kDa); 1. Cell lysate; 2. Supernatant; 3. Pellet; 4. Unbound protein; 5-6.

Wash; 7. Elution; 8. Diluted elution protein...... 91

Figure 4.6.3 A) Scheme for the generation of N001 in one pot reaction. B) Scheme for the

generation of N3001 in one pot reaction...... 92

Figure 4.6.4 LacZ digestion of N001 over time, reaction monitored by HPLC-ELSD. A) N001

control. B) N211 control. C) N000 control. 0) Negative control no enzyme. 1)Reaction 0

min. 2) Reaction 1 h. 3) Reaction 2 h. 4) Reaction 3 h, 85% conversion to intermediate

product. 5) Reaction 4 h. 6) Reaction 5 h. 7) Reaction 6 h. 8) Reaction 7 h. 9) Reaction 8

h. 10) Reaction ov, 47% conversion to intermediate product, and 53% conversion to

N000 product...... 94

Figure 4.6.5 LacZ digestion of N3001 over 4 days, each intermediate product was purified and

confirmed by MALDI-MS. I) N3001 II) Intermediate product without 1 galactose residue

(N3111). III) Intermediate product without 2 galactose residues (N3361). IV) Completely

digested product (N000)...... 95

Figure 4.6.6 Scheme for the separation of N-glycan isomers from digestion of N001 with LacZ

using FUT8 and Gn-T V...... 96

Figure 4.6.7 Scheme for LacZ digestion of N3001 and identification of possible isomer

intermediate using FUT8 and Gn-T V. Reactions were monitored by MALDI-MS. A.

N3001 -1 galactose residue possible isomers. B. N3001 -2 galactose residues possible

isomers. Red box indicates the isomers identified from the MALDI-MS results (See

pg.93 §4.6.11)...... 97

Figure 4.6.8 Gn-T V catalyzed reaction for bi- and tri- antennary N-glycans...... 99

1 INTRODUCTION

1.1 Purpose

The goal of this dissertation is to study and apply human glycosyltransferases (hGT) substrate specificity to N-glycan synthesis. Five hGT were expressed, purified and their activities assayed. Two of them were used in synthetic schemes for the extension and separation of asymmetric N-glycan isomers. hGTs are enzymes involved in the glycan biosynthesis. The natural overexpression of some of these enzymes such as FucT8 and Gn-T V can contribute to the development, progression, malignancy, recurrence, invasive and metastatic properties of cancer cells, among others. 1-5

The following are the hGTs that were expressed: 1. a-1,3-mannosyl-2-b-N- acetylglucosaminyltransferase (Gn-T I), 2. a-1,6-mannosyl-2-b-N-acetylglucosaminyltransferase

(Gn-T II), 3. b-1,4-mannosyl-2-b-N-acetylglucosaminyltransferase (Gn-T II), 4. a-(1,6)- fucosyltransferase (FUT8), and 5. b-1,6-mannosyl-2-b-N acetylglucosaminyltransferase (Gn-T

V). The substrate specificity studies on these enzymes have and will continue to be applied towards the development of libraries of structurally well-defined N-glycans, which is an urgent scientific need for quantification and characterization of N-glycans on proteins, rapid analysis of carbohydrate binding proteins (CBPs), and the development of diagnostics and therapeutics for many diseases. This research area is challenging due to the complexity, diversity and low abundance of glycan structures. The availability of structurally well-defined N-glycan libraries is essential to help solve these issues. However, only a small number of N-glycans are currently commercially available, which is a disadvantaged for glycobiology research.

Our group developed an N-glycan library, which established an efficient chemo- enzymatic strategy, named Core Synthesis/Enzymatic Extension (CSEE), for rapid production of

12 diverse N-glycans.6 Taking advantage of this method asymmetric core-fucosylated N-glycans

(CF-glycans) can be chemo-enzymatically synthesized in order to extend our existing library.

The generation of well-defined core fucosylated N-glycan structures are useful as standards for quantitative glycomics, analysis of glycan structures, and as antigens for the production of diagnostic antibodies.7 Taking advantage of well-studied substrate specificity of two out of the five hGTs expressed (FUT8 and Gn-T V) enzymatic separation of asymmetric N-glycan isomers was accomplished. Insight on the substrate specificity of these important hGTs is important for future development of N-glycan synthesis.

In the following Chapters this dissertation will cover: Chapter 1 a brief introduction into glycosylation. Chapter 2 the substrate specificity of FUT8 and chemo-enzymatic synthesis of core fucosylated asymmetric N-glycans published on Organic and Biomolecular Chemistry.

Chapter 3 a review on FUT8: from biochemistry to synthesis of core-fucosylated N-glycans accepted for publication on Pure and Applied Chemistry. Chapter 4 is a manuscript in progress for the enzymatic separation of asymmetric N-glycan isomers using FUT8 and Gn-T V substrate specificity. Additional information on Gn-T I, II, and III can be found in Appendix A.

1.2 Introduction

Post-translational modifications are very important since they influence folding, stability, signaling, transport and life span of proteins. 8, 9 There are four types of glycosylated proteins known as glycoproteins: O-linked, N-linked, glycosaminoglycans, and glycosylphosphatidylinositol (GPI)-anchored proteins(Figure 1.2.1).10

Chondroitin/dermatan sulphate P S 4S 4S 4S 4S Proteoglycans S Symbols:

Gal S

Heparan sulphate Glc S S 6S 6S 6S 6S 6S S N-linked chains S S Man NS NS 2S NS S O-linked chains L-Fuc S Glycosphingolipids GlcNAc S S/T Neu5Ac P S/T N S/T N Neu5Gc 3S Glycoproteins S/T

Figure 1.2.1 Glycosylation types.

Here we will only focus on N-linked glycosylation. There are three types of N-glycans in a mature glycoprotein: oligomannose, complex, and hybrid (Figure 1.2.2). Each N-glycan

8 contains the common core Man3GlcNAc2Asn. Research has been done previously in order to determine the glycosylation site Asn-X-Ser/Thr.8, 11 Some amino acids have been found to inhibit glycosylation at the X position such as Pro.

a6 a6 a6

a2 a2 a2 b4 b4 b4 a2 a2

a2 a3 a6 b2 b2 b2 a3 a6

a3 a6 a3 a6 a3 a6 b4 b4 b4

b4 b4 b4 a6 b b b N N N High Complex Hybrid mannose

Figure 1.2.2 N-glycan structures found in mature glycoprotein.

1.3 Glycosylation importance

Glycosylation of proteins often aids with folding and stability of secreted proteins.

Oligosaccharides are important in many recognition, signaling and adhesion events within and between cells. Glycans have an important role in the biological function of proteins such as molecular stability, solubility, in vivo activity, serum half-life, and immunogenicity.10, 12-17 One important aspect of protein glycosylation is the function of the sialic acid component of the glycan. Sialic acids on carbohydrates form highly hydrophilic structures, therefore increasing the solubility of the proteins by shielding hydrophobic residues. They also have the ability to extend the half-life of proteins, which is very important for protein therapeutics. Glycans can also reduce the susceptibility to proteolysis, and the sensitivity to thermal denaturation as it has been shown for glycosylated interferon beta (IFN-b).10 Another advantage with glycosylation is the potential to minimize the immune response that might be provoked by the generation of neutralizing antibodies against the engineered protein.10 Glycosylation is likely to reduce the immune response by increasing solubility and reducing aggregation. It can also inhibit the antibody binding by “shielding” the protein sequences from the immune system. The glycan shielding protects the engineered protein sequence from immune surveillance.10

1.4 N-Glycosylation site

The commonly known N-glycosylation sequon known as Asn-X-Ser/Thr is many times poorly glycosylated or not glycosylated at all.8 This inefficiency in N-linked glycosylation led scientist to elucidate more on the required sequon for N-glycosylation. Using cassette mutagenesis approach a group was able to identify a more specific N-glycosylation sequon.

Their proposed sequon adds an additional term Y to the known one and examines amino acids that are favorable at the X and Y positions for the increase in N-glycosylation efficiency. 11

From previous reports it is known that this specific sequon can be improved by replacing Leu38 with more favorable amino acid as well as replacing Ser39 with Thr can improve the glycosylation. The conclusion from these studies showed that position Y amino acid is actually important in order to increase the core glycosylation efficiency, especially when Ser is the hydroxyl amino acid. From their results they were able to determine that Pro, Trp, and Glu inhibit glycosylation when present at the Y position just like they do when present at the X position. They also suggest that large side groups may block how accessible the oligosaccharyltransferase and/or dolichol oligosaccharide donors are. It may also form an unfavorable local protein conformation. The effect of charge amino acids at the X and Y position is complex. While Glu inhibits glycosylation, Asp only has some inhibitory effect not as notable as Glu. Positively charged amino acids Arg appears to be the most favorable at Y position compared to His or Lys. The most favorable amino acids at both positions X and Y are

Thr, Ser and Cys. From the study they suggest that probably the most favorable amino acids with side chains containing hydroxy or sulfhydryl can facilitate the catalytic reactions that involve Asn and Ser/Thr positions.11

1.5 N-Glycosylation difference

The N-glycan pathway is very conserved including the generation and transfer of

Glc3Man9GlcNAc2 from dolichol phosphate to asparagine residues on newly synthesized proteins. Even though, glycans are found in all organism there is a lot of diversity in the structure and expression among all organism. N-glycan pathway is found throughout all eukaryotes, but recently there is data indicating that some bacteria such as Campylobacter jejuni can also express similar N-glycan structures. Glycans expressed by most free-living eubacteria and archaea have very little in common structurally with those of eukaryotes. 18

1.6 Protein glycosylation pathway

It has been established that the major site for protein glycosylation is the endoplasmic reticulum (ER) followed by the golgi apparatus. These two cell organelles form an endomembrane assembly line on which membrane-bound glycosyltransferases and glycosidases process the N-glycans attached to Asn key glycosylation site of the protein as they move towards their final destination (secretion, cell surface or lysosome).19 The Asn-GlcNAc bond is primarily formed on the nascent polypeptide while it is assembled into the ER by a membrane-bound ribosome. Complex N-glycans come from Glc3Man9GlcNAc2-pyrophosphate-dolichol, a lipid linked oligosaccharide (LLO), by a very extensive N-glycan processing pathway.8, 19 The synthesis of complex N-glycan proteins can be divided into three stages. First is the formation of

Glc3Man9GlcNAc2-pyrophosphate-dolichol between the cytoplasm and the rough ER lumen.

The second stage takes place in the ER and is the transfer of Glc3Man9GlcNAc2 from pyrophosphate-dolichol to the Asn key glycosylation site on the nascent protein (Asn-X-Ser/Thr-

Y) which is catalyzed by Oligosaccharyltransferase (OST). The third stage takes place in the cis, medial, and trans sections of the golgi apparatus. This stage is mostly found in eukaryotes since it requires a transfer of a GlcNAc residue with a b-1,2 linkage to the Man-a-1,3 branch. This reaction is catalyzed by Gn-T I and followed by mannosidase II and Gn-T II to yield hybrid or complex N-glycans from oligomannose N-glycans (Figure 1.6.1). 8, 19

OST Glycoprotein ER folding P N N N N P Cytosol

Gn-T I Cis- golgi

N N N

Gn-T III N Gn-T II Medial- Gn-T V golgi N N N N

FUT8

Trans- golgi ST6GalT GalT N N N N Secretion or movement to plasma membrane

Figure 1.6.1 Summary of processing and maturation of an N-glycan protein from the ribosome through the ER and Golgi apparatus to its final destination.8

After the transfer of Glc3Man9GlcNAc2 glycan to Asn on the nascent protein, glucosidases in the ER remove the three glucose residues, and mannosidases remove the mannose residues. Glucosidases and mannosidase are associated with the folding of the glycoprotein, which is aided by a couple of lectins that will determine if the glycoprotein goes to the Golgi or is degraded. If the glycoprotein is misfolded the ER degradation-enhancing α-

18 mannosidase I–like protein (EDEM) will bind to mannose residues and transport it to the cytoplasm for degradation. The removal of additional mannose residues takes place at the cis-

Golgi apparatus until the core N-glycan is formed (Man5GlcNAc2Asn). The formation of the bi- anternnary N-glycan starts at the medial-Golgi with the action of Gn-T I adding a GlcNAc at the a-1,3 mannose followed by α-Mannosidase II to remove two mannoses. Gn-T II then adds another GlcNAc at the a-1,6 mannose branch (Figure 1.6.1). The bi-antennary N-glycan can be further extended by addition of fucose, galactose, and sialic acid to generate a complex N-glycan.

Complex N-glycans can also have addional branching and extesion.8

1.7 Therapeutic N-linked glycoproteins

Many important therapeutic proteins are glycosylated including Erythropoetin (EPO), granulocyte macrophage colony stimulating factor (GM-CSF) and some monoclonal antibodies

(mAb) such as Etanercept, Infliximab and Rituximab.9 Therapeutic drugs glycosylation is very important as it was previously discussed. One example of the importance is EPO. Experiments performed on EPO glycosylated analogs have shown a direct relationship between the number of glycan chains and the increase in in vivo activity. The increase in biological efficiency of glycosylated proteins can be due to the increase in serum half-life or circulating residence time.10

The increase in biological activity efficiency was shown in recombinant human erythropoietin

(rHuEPO). Darbepoetin alfa (DA) a hyperglycosylated analogue of rHuEPO, shows a three fold increase in serum half-life when compared to rHuEPO. 10

Rituximab is another great example of N-glycosylation importance for protein drugs biological activity enhancement. This protein drug is a monoclonal antibody (mAb) against

CD20. mAbs are one of the fastest growing classes of pharmaceutical products. They are used to treat conditions such as cancer, infectious diseases, allergies, inflammation, and auto-immune

19 diseases. Rituximab was approved in 1997 by the FDA as cancer therapy for the treatment of malignant lymphomas. Rituximab is made up of 2 light chains containing 213 amino acids and 2 heavy chains with 451 amino acids. It has 12 intrachain disulfide linkages and 4 interchain disulfide linkages. Rituximab only contains one glycosylation site on both heavy chains at

Asn297 .20,21 The efficiency of this mAb is greatly influenced by the diversity and abundance of each glycan structure. Mass spectroscopy (MS) is an essential tool widely use in the pharmaceutical industry for the characterization of mAbs in order to gain FDA approval. MS is useful for determination of molecular weight of intact proteins, glycosylation location, glycan structure abundance, and amino acid sequence confirmation.20 Therefore, efficient access to well-defined glycan structures as strandards in glycomic research is very useful in the pharmaceutical industry.

1.8 References:

1. K. Kamio, T. Yoshida, C. Gao, T. Ishii, F. Ota, T. Motegi, S. Kobayashi, R. Fujinawa, K. Ohtsubo, S. Kitazume, T. Angata, A. Azuma, A. Gemma, M. Nishimura, T. Betsuyaku, K. Kida and N. Taniguchi, Biochem Biophys Res Commun, 2012, 424, 112-117. 2. T. Fukuda, H. Hashimoto, N. Okayasu, A. Kameyama, H. Onogi, O. Nakagawasai, T. Nakazawa, T. Kurosawa, Y. Hao, T. Isaji, T. Tadano, H. Narimatsu, N. Taniguchi and J. Gu, J Biol Chem, 2011, 286, 18434-18443. 3. E. Miyoshi, K. Noda, Y. Yamaguchi, S. Inoue, Y. Ikeda, W. Wang, J. H. Ko, N. Uozumi, W. Li and N. Taniguchi, Biochim Biophys Acta, 1999, 1473, 9-20. 4. H. Imai-Nishiya, K. Mori, M. Inoue, M. Wakitani, S. Iida, K. Shitara and M. Satoh, BMC Biotechnol, 2007, 7, 84. 5. X. Wang, J. Chen, Q. K. Li, S. B. Peskoe, B. Zhang, C. Choi, E. A. Platz and H. Zhang, Glycobiology, 2014, 24, 935-944. 6. L. Li, Y. Liu, C. Ma, J. Qu, A. D. Calderon, B. Wu, N. Wei, X. Wang, Y. Guo, Z. Xiao, J. Song, G. Sugiarto, Y. Li, H. Yu, X. Chen and P. G. Wang, Chemical science, 2015, 6, 5652-5661. 7. S. Serna, S. Yan, M. Martin-Lomas, I. B. Wilson and N. C. Reichardt, Journal of the American Chemical Society, 2011, 133, 16495-16502. 8. R. D. Cummings and J. D. Esko, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009.

9. M. Kuhlmann and A. Covic, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2006, 21 Suppl 5, v4-8. 10. A. M. Sinclair and S. Elliott, Journal of pharmaceutical sciences, 2005, 94, 1626-1635. 11. J. L. Mellquist, L. Kasturi, S. L. Spitalnik and S. H. Shakin-Eshleman, Biochemistry, 1998, 37, 6833-6837. 12. R. A. Dwek, Chemical reviews, 1996, 96, 683-720. 13. B. Imperiali and S. E. O'Connor, Current opinion in chemical biology, 1999, 3, 643-649. 14. A. Helenius and M. Aebi, Science, 2001, 291, 2364-2369. 15. A. Helenius and M. Aebi, Annual review of biochemistry, 2004, 73, 1019-1049. 16. A. Varki and J. B. Lowe, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 17. Y. Y. Zhao, M. Takahashi, J. G. Gu, E. Miyoshi, A. Matsumoto, S. Kitazume and N. Taniguchi, Cancer science, 2008, 99, 1304-1310. 18. R. D. Cummings and J. D. Esko, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 19. H. Schachter, Glycoconjugate journal, 2000, 17, 465-483. 20. M. Samonig, C. Hber and K. Scheffler, Thermo Fisher Scientific Application Note 591, 1-11. 21. V. H. Pomin, Unravelling Glycobiology by NMR Spectroscopy, 2012.

2 SUBSTRATE SPECIFICITY OF FUT8 AND CHEMOENZYMATIC SYNTHESIS

OF CORE-FUCOSYLATED ASYMMETRIC N-GLYCANS

The work presented in this chapter is reproduced with some modification as to fit the format with permission from journal: “Substrate Specificity of FUT8 and Chemoenzymatic

Synthesis of Core-fucosylated Asymmetric N-glycans.” Angie D. Calderon, Yunpeng Liu, Xu

Li, Xuan Wang, Xi Chen, Lei Li and Peng G. Wang. Organic and Biomolecular Chemistry.

(2016) 14 (17) 4027-3.

2.1 Abstract

Substrate specificity studies of human FUT8 using 77 structurally-defined N-glycans as acceptors showed a strict requirement towards the a1,3-mannose branch, but a great promiscuity towards the a1,6-mannose branch. Accordingly, a chemo-enzymatic strategy was developed for efficient synthesis of core-fucosylated asymmetric N-glycans.

2.2 Introduction

Asparagine-linked glycosylation (N-glycosylation) represents the most prevalent and structurally varied post-translational modification of eukaryotic proteins. Tremendous advances in glycobiology have demonstrated that asparagine-linked oligosaccharides (N-glycans) can modulate protein’s structure and function in many ways. For example, they were found to play roles in a variety of biological processes, including cell adhesion, pathogen infections, immune responses and tumor metastasis.1-3 On the other hand, N-glycans can influence proteins biosynthesis, folding, stability, antigenicity and immunogenicity.3-6 N-Glycans found in nature possesses inherent complexity and diversity, due to variable connectivity of monosaccharide building blocks as well as additional modifications on the basic scaffolds. Core-fucosylation is a major N-glycan modification in eukaryotic glycomes.

In mammalian cells, core-fucosylation is exclusively found as a1,6-fucosylation of the innermost N-acetyl-glucosamine (GlcNAc) residue of N-glycans. Many glycoproteins are known to be core-fucosylated, and accumulating data indicate that this type of modification regulates the functions of certain glycoproteins. For example, depletion of N-glycan core-fucosylation in human

IgG1 enhanced its antibody-dependent cytotoxicity (ADCC) activity to 50 - 100 folds.7, 8 It was shown that core-fucosylation regulates the functions of immunoglobulin by altering its physicochemical properties.9 Another most recent example is that in humoral immune response, core-fucosylation of IgG B cell receptor was shown to mediate antigen recognition and cell signal transduction.10 Alteration of core-fucosylation is frequently found to be closely related to human diseases including liver cancer, pancreatic cancer, lung cancer, breast cancer, etc.11, 12 A well- known example is a-fetoprotein, which was found highly core-fucosylated specifically in hepatocellular carcinoma, but not other liver diseases.13-16 In addition, E-cadherin, a glycoprotein correlated with cancer metastasis, was found to be core-fucosylated in highly metastatic lung cancer cells but absent in low metastatic ones.17 Compare to controls, increased core-fucosylated

N-glycans and decreased non-fucosylated ones were also detected in the clinical ovarian cancer biomarker CA125.18 Moreover, core-fucosylation is pivotal in development, and the loss of such

N-glycan modification leads to severe phenotypes such as growth retardation, emphysema-like changes in lung, even death.19 All these evidences strongly suggest that core-fucosylation plays vital roles in various biological processes and events.

FUT8 is the only enzyme responsible for core-fucosylation in mammals, which catalyzes the transfer of an L-fucose residue from GDP-b-L-fucose (GDP-Fuc) onto the innermost GlcNAc of N-glycan to form an a1,6-linkage (Figure 2.2.1).19, 20 It has been reported that FUT8 is highly expressed in brain tissue (where core-fucosylated glycoproteins were found abundant), human

23 blood platelets, cancer tissues, and other tissues during certain pathological processes. Previous substrate specificity study suggested that FUT8 requires both a1,3-mannose branch terminal

GlcNAc residue and reducing end b-configuration of the acceptor.21-24 In addition, galactosylation of the GlcNAc residue at a1,3-mannose branch and bisecting of N-glycans prevent FUT8 activity.21, 22 Most recently, we and others showed that both human and Caenorhabditis elegans

FUT8 can fucosylate asymmetric N-glycans (Figure 2.2.1, N211 and N212), indicating that they may have a much relaxed acceptor substrate preference towards the a1,6-mannose branch.25, 26 A comprehensive study is necessary to confirm such specificity, and structurally defined N-glycans that we synthesized previously26 represent perfect substrate candidates for test.

Given the significance, FUT8 and core-fucosylation have been extensively investigated in terms of glycomics and in vivo biological functions, while in depth molecular elucidation is largely limited by the unavailability of structurally well-defined N-glycans with core- fucosylation. In the past decade, a few chemical and enzymatic methodologies were developed for the preparation of such molecules. Unverzagt, used a modular chemical synthesis strategy where the a1,6-fucose was attached in the last step.27-29 On the other hand, Huang applied a pre- activation-based one-pot chemical strategy to quickly assemble a dodecasaccharide N-glycan structure.30 Enzymatically, Reichardt used C. elegans FUT8 to core-fucosylate chemically prepared simple N-glycan structures (Figure 2.2.1, N000 and N010),31 while Wang applied glycosynthases to conjugate chemically prepared glycan oxazolines and Fuca1,6-GlcNAc to form homogeneous core-fucosylated N-glycans and glycoproteins.32, 33 In all cases, only simple or symmetric core-fucosylated N-glycans were prepared, and an efficient strategy for rapid access of more complicated and asymmetric ones is yet to be developed. In this work, human

FUT8 was overexpressed and purified, detailed acceptor substrate specificity was investigated

24 towards 77 structurally defined N-glycans and derivatives. Accordingly, a chemoenzymatic strategy was developed for efficient synthesis of core-fucosylated asymmetric N-glycans.

Figure 2.2.1 FUT8 catalyzed reaction and substrate specificity of human FUT8 using 77 N-glycans as potential acceptors (see Figure 2.6.3 for the full list of N-glycans, Table 2.6.3.1 for conversion percentage towards each glycan). The average conversion percentage of three replicates for each acceptor was shown.

2.3 Results and Discussion

Human FUT8 was chosen for substrate specificity study and synthetic purpose.

Heterogeneous expression was achieved via a Titerless Infected-cell Preservation and Scale-up

(TIPS) approach34 using a baculovirus system (see http://glycoenzymes.ccrc.uga.edu for system and construction details) provided by Dr. Jarvis from the University of Wyoming. After one-step

Ni-Sepharose Excell (GE Healthcare) affinity purification, recombinant FUT8 with a purity of over 90% (Figure 2.6.1) was obtained in a scale of 0.42 mg/L of cultures. Substrate specificity study was then performed in reactions with a total volume of 20 µL containing an acceptor N- glycan (0.3 mM), donor GDP-Fuc (1 mM), MES buffer (100 mM, pH 7.0) and 50 µg/mL of the recombinant human FUT8. After 4 h of incubation at 37 °C, the reactions were quenched by the

25 addition of equal volumes of ice cold ethanol, followed by analysis using mass spectrometry (MS) and high performance liquid chromatography (HPLC) monitored by an evaporative light scattering detector (ELSD) (See pg. 33 § 2.6.3).

Consistent with previous reports21-24 as shown in Figure 2.2.1, FUT8 could core-fucosylate

N-glycans with an agalactosylated and unprotected GlcNAc residue on the a1,3-mannose branch

(N000, N010, N020, N030, N110, and N211-N215), but not those either galactosylated or with protected GlcNAc residue on the branch. Among these N-glycans, FUT8 showed the highest activity towards biantennary complex N-glycan structures terminated with GlcNAc at the non- reducing end of both a1,3- and a1,6-mannose branches N000 (91.3% conversion) and slightly decreased activities towards N110 (63.4% conversion) and N211-N213 (67.8% to 70.4% conversion), indicating that galactosylation with or without additional sialylation or chemical protection of GlcNAc on the a1,6-mannose branch can be well tolerated. The addition of an a1,3- linked fucose to the terminal N-acetyllactosamine (LacNAc) or sialyl LacNAc structures on N211 and N212 to form the Lewis x and sialyl Lewis x structures on the a1,6-mannose branch of N214

(40.1%) and N215 (34.8%), respectively, further decrease the efficiency for FUT8-catalyzed core fucosylation. Quite interestingly, FUT8 was found to be able to glycosylate N04, an N-glycan without a a1,3-mannose branch, even though with a neglectable percentage conversion (5.26%).

The result was confirmed by milligram scale synthesis and product N604 purification followed by mass spectrometry (MS) and proton nuclear magnetic resonance (1H NMR) characterization (See pg. 53 § 2.6.6). Overall, our substrate specificity study using a library of 77 structurally defined

N-glycans confirmed that FUT8 has a quite strict requirement towards the a1,3-mannose branch, and suggested a very relaxed preference towards the a1,6-mannose branch.

Figure 2.3.1 Two N-glycans (m/z) identified from CA125 and possible N-glycans (black squared). Red dash squared N-glycan structures were synthesized in this study.

With the information about substrate specificity of FUT8 in hand, we were ready to design effective routes for synthesizing asymmetric N-glycans with core-fucosylation which have been frequently found in the glycomes of mammalian cells, especially on disease related proteins.35-37

For example, putative structures of m/z 2605 and 2780 found on human ovarian biomarker glycoprotein CA125 were determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and collision activated dissociation-tandem mass spectrometry (CAD-

MS/MS).38 Nevertheless, a few possible isomers (Figure 2.3.1) were not ruled out. To access such asymmetric N-glycans with core-fucosylation which can be used as glycan standards, a facile chemoenzymatic synthetic strategy was developed (Figure 2.3.2 and

Figure 2.3.3).

Similar to our core synthesis/enzymatic extension strategy (CSEE) for the preparation of asymmetric N-glycans,26 we envisaged N6111 and N6211 as two key intermediates for the synthesis of core-fucosylated ones. As illustrated in Figure 2.3.2A, to obtain intermediate N6111,

27 we chose chemically prepared N-glycan N110 26 as a starting point, which was shown to be an well tolerated substrate of human FUT8 in the substrate specificity study described above. Briefly, in a 2.5 mL reaction system containing 100 mM MES buffer (pH 7.0), 7.2 mg (5 µmole, 2 mM) of N110 was incubated at 37 °C with GDP-Fuc (4 mM), recombinant FUT8 (0.2 mg/mL), and 10

U/mL of thermosensitive alkaline phosphatase (FastAP, ThermoFisher) to drive the reaction forward by digesting byproduct GDP. Every other hour, 2 µL of the reaction mixture was withdrawn for analysis. MALDI-MS analysis showed a peak at m/z = 1611.5695, corresponding

+ to N6110 [M + H] . Meanwhile, on the HPLC-ELSD profile, a new peak (TR = 6.99 min) was observed, of which the area increased while that of the peak corresponding to N110 (TR = 6.06 min) decreased. After N110 was completely fucosylated (8 h incubation), the reaction was freeze- quenched at -80 °C for 30 min, and the mixture was concentrated to 300 mL for HPLC purification with a water/acetonitrile gradient elution (See pg. 36 § 2.6.4 for details) to provide 6.9 mg of

N6110 (87% yield). The purified N6110 was then galactosylated to afford N6111a by bovine b1,4- galactosyltransferase (b4GALT1) as described previously.26 In this case, only the GlcNAc residue on the a1,3-mannose branch was galactosylated, as the other one on the a1,3-mannose branch was protected by peracetylation. After HPLC purification, the GlcNAc residue on the a1,6-mannose branch of N6111a was de-acetylated with 3% of ammonium hydroxide to afford 6.5 mg (after

HPLC purification) of key intermediate N6111 (80% yield over 3 steps).

Figure 2.3.2 Chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans from N110 (A), and MALDI-MS analysis of purified glycans (B). Reaction conditions: a) FUT8, GDP-Fuc, FastAP; 2+ b) b4GALT1, UDP-Gal, FastAP, Mn ; c) 30% ammonium hydroxide : H2O (1 : 10), 6 h; d) PmST1m, CMP-Neu5Ac; e) Pd2,6ST, CMP-Neu5Ac; f) Hpa1,3FT, GDP-Fuc, FastAP, Mn2+. FUT8, human a1,6-fucosyltransferase; b4GALT1, bovine b1,4-galactosyltransferase; PmST1m, Pasteurella multocida a2,3-sialyltransferase 1 mutant E271F/R313Y; Pd2,6ST, Photobacterium damselae a 2,6- sialyltransferase; Hpa1,3FT, c-terminal 66 amino acids truncated Helicobacter pylori a1,3- fucosyltransferase; FastAP, thermo-sensitive alkaline phosphatase.

With N6111 in hand, core-fucosylated asymmetric N-glycans N6122, N6123 and N6244 were prepared in a step-wise manner similar to that of the non-core-fucosylated ones.26 Besides b4GALT1, 3 bacterial glycosyltransferases (GTs) were employed in the synthesis, including a sialidase activity reduced mutant (E271F/R313Y) of Pasteurella multocida a2,3-sialyltransferase

1 (PmST1m)39 to catalyze the formation of a2,3-sialosides, a Photobacterium damselae a2,6- sialyltransferase (Pd2,6ST)40 to catalyze the formation of a2,6-sialosides, and a C-terminal 66 amino acids truncated version of Helicobacter pylori a1,3-fucosyltransferase (Hpa1,3FT)26 to fucosylate branched GlcNAc residues (Figure 2.3.2A). All glycans were purified by HPLC after each glycosylation step and characterized by MALDI-MS (Figure 2.3.2B). N-Glycan purity was confirmed by HPLC-ELSD (See pg. 37 § 2.6.5). It is worth to mention that none of the 4 GTs showed altered activity towards core-fucosylated N-glycan substrates compare to that for non- core-fucosylated ones, indicating that the a1,6-core fucose has negligible effect on acceptor glycan interaction with the GTs. More core-fucosylated asymmetric N-glycans can thus be readily synthesized from N6111 following previously reported routes.26

Figure 2.3.3 Enzymatic synthesis of core-fucosylated N-glycans starting with N210. (See Figure 2.3.2A) for detailed description of each step.

In contrast to N110 with a per-acetylated GlcNAc at the a1,6-mannose branch which is a suitable acceptor for FUT8, N210 with a per-acetylated GlcNAc at the a1,3-mannose branch is not a suitable substrate for FUT8. Therefore, a key intermediate N6211 was needed for further enzymatic extension. It was synthesized from N210 by b1,4-galactosylation of the non-protected

GlcNAc on the a1,6-mannose branch for the formation of N211 followed by deprotection of the

GlcNAc on the a1,3-mannose branch and FUT8-catalyzed core-fucosylation of the asymmetric

N-glycan (

Figure 2.3.3). Starting from N6211, desired N-glycans N6222, N6223 and N6144 were then enzymatically synthesized in a step-wise manner as described above (

Figure 2.3.3). The purified products were characterized by MALDI-MS, 1H NMR (See pg.

37 § 2.6.5), 1H NMR (See pg. 54 § 2.6.7), and HPLC-ELSD (See pg. 37 § 2.6.5).

2.4 Conclusion

In conclusion, the available of previously synthesized 77 structurally-defined N-glycans allowed detailed substrate specificity study of a recombinant human FUT8. Results showed a quite strict requirement of FUT8 towards the a1,3-mannose branch but a great promiscuity towards the a1,6-mannose branch. Interestingly, human FUT8 can also core-fucosylated an N-glycan without the a1,3-mannose branch, which was not observed before. In addition, a facile chemo-enzymatic strategy was developed and employed for the efficient synthesis of a panel of core-fucosylated asymmetric N-glycans. These well-defined structures are valuable standards and probes for glycomics analysis and elucidating functions of glycan-binding protein.

2.5 Acknowledgements

We are grateful to Dr. Donald Jarvis from the University of Wyoming for kindly providing the Sf9 cells and FUT8 baculovirus stock. This work was supported by National Institutes of Health

(U01GM0116263 to P.G. Wang and L. Li) and National Cancer Institute (Contract SBIR:

HHSN261201500021C to Chemily, LLC).

2.6 Experimental

2.6.1 Materials and enzymes

b1,4-galactosyltransferase from bovine milk (B4GALT1) was purchased from Sigma.

Thermosensitive Alkaline Phosphatase (FastAP) was purchased from Thermo Scientific. N,N’- diacetyl-chitobiose (N01) was purchased from Sigma, and N-glycans N02, N03, N04, N05, N06 were purchased from V-labs (Covington, LA). Other enzymes including double mutant

E271F/R313Y from Pasteurella multocida a2,3-sialyltransferase 1 (PmST1m)39, a2,6- sialyltransferase from Photobacterium damslae (Pd2,6ST)40, C-terminal 66 amino acids truncated

Helicobacter pylori a1,3-fucosyltransferase (Hpa1,3FT)41, CMP-sialic acid synthetase from N. meningitidis (NmCSS)42 were expressed and purified as previously described. Enzymes were then desalted against 50 mM Tris-HCl, 100 mM NaCl, and 50% glycerol, and stored at -20 °C for long term use. Sugar nucleotides uridine 5’-diphospho-galactose (UDP-Gal)43, cytidine 5’- monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)42 and guanosine 5’-diphospho-L-fucose

(GDP-Fuc)44 were prepared as described previously.

2.6.2 Expression and purification of human FUT8

2.6.2.1 Cryopreservation and maintenance of Baculovirus infected Sf9 cells

The 50 µL of P1PP1 baculovirus stock and Sf9 cells for the expression of the FUT8 were kindly provided by Dr. Donald Jarvis from University of Wyoming. For the Titerless Infected- cells Preservation and Scale-up (TIPS) expression method34, a primary stock of cryopreserved infected cells BIIC was prepared by infecting 25 mL of Sf9 cells at a concentration of 1×106 C/mL

32 with 25 µL of untitered liquid P1PP1 baculovirus stock (an estimated MOI of approximately 3 pfu/cell). The cells were cryopreserved when the average diameter increases by 20-30% of their pre-infected diameter and the viability was above 95%. Sf9 cells were grown and infected at 28

°C and 150 rpm in complete TNM-FH medium [TNM-FH medium (Sigma-Aldrich), supplemented with 10% Fetal Bovine Serum (FBS) (BioWest), 0.5% antibiotic-antimycotic

(Sigma-Aldrich), and 0.1% Pluronic-F68 (Invitrogen)]. The cells were cryopreserved at a density of 1×107 C/mL in liquid nitrogen and complete TNM-FH medium and 10% DMSO (Sigma-

Aldrich). Cell density was determined by hemocytometer counts, and the cell viability was evaluated by Trypan Blue staining.

2.6.2.2 FUT8 expression and purification

Sf9 cells were infected at a cell density of 1×106 C/mL. The baculovirus titer was estimated using insect cell density at time of infection and a conservative cellular baculovirus maximum production rate of 100 pfu/cell for 1×106 C/mL meaning 1×108 pfu/mL. BIIC stock was thawed in a 37 °C water bath and diluted 1:100 into cell-free medium, then 10 mL of infected cell-free medium was added to 1 L of uninfected cells (1:1000 dilution)34. The medium was collected after

72 h of expression followed by purification. The purification of the secreted protein was accomplished by diluting the medium 1:1 with equilibration buffer (50 mM Tris-HCl, pH 7.5, 0.5

M NaCl) and loading to 2 mL of pre-equilibrated Ni-Sepharose Excell (GE Healthcare)45. The column was rinsed using 200 mL of wash buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 50 mM imidazole), and finally eluted with 5 mL of elution buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl,

250 mM imidazole). The enzyme was desalted and concentrated using Amicon YM-30 ultrafiltration membrane (Millipore) against desalting buffer (50 mM Tris-HCl pH 7.5). Purified

FUT8 was confirmed by SDS-PAGE (>90% pure) and western blot (Figure 2.6.1). Using TIPS

33 method 0.42 mg of human FucT8 were purified from 1 L cultures as determined using Bradford method. The activity of recombinant FUT8 was confirmed by using N000 as a substrate, monitored by MS (Figure 2.6.2).

Figure 2.6.1 SDS-PAGE and western blot analysis of FUT8. 1) SDS-PAGE of medium after overexpression of FUT8; 2) SDS-PAGE of purified FUT8; and 3) western blot analysis of purified FUT8 using anti-His antibody as the primary antibody. BSA, a major protein component of Fetal Bovine Serum used in the culture medium, has similar molecular weight as that of FUT8.

4 x10 1485.5385 2.0

Intens. [a.u.] Intens. 1.5

1.0

0.5 1339.4568

0.0 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z Figure 2.6.2 MALDI-MS analysis of FUT8 catalyzed reaction using N000 as substrate. Majority of N000 (molecular weight 1316.4865, found M+Na 1339.4568) was converted into core-fucosylated one (molecular weight 1462.5444, found M+Na 1485.5385). 2.6.3 Substrate specificity study of FUT8

A detailed substrate specificity study of FUT8 was performed using 77 chemically or chemo-enzymatically synthesized, or commercially available N-glycans (with free reducing end) as acceptors (Figure 2.6.3). Reactions were performed in a 96 well PCR plate, with each well (20

µL total volume) contains one N-glycan substrate (0.3 mM, 2.5 µg to 15.1 µg), GDP-Fuc (1 mM),

FUT8 (0.05 mg/mL) and MES buffer (100 mM, pH 7.0). Reactions were incubated at 37 °C for 4 h in an Eppendorf thermocycler (Mastercycler Pro), followed by enzyme inactivation at 95 °C for

20 min. A negative control was also set up using N000 as substrate without enzyme. After centrifugation (13,000 g for 10 min), the supernatant was analyzed by HPLC using an analytical

Waters XBridge BEH amide column (130 Å, 5 µm, 4.6 x 250 mm), detected by an evaporative light scattering detector (Shimadzu ELSD-LTII). Gradient elution (%B: 75– 60% for glycans with less than 9 monosaccharides, 70– 55% for glycans with 9 or more monosaccharides) was performed with Solvent A (100 mM ammonium formate, pH 3.4) and Solvent B (Acetonitrile).

The percent yields (average of three replicated reactions, see (Table 2.6.3.1) were calculated as %

= Product peak area/ (Product peak area + Substrate peak area) × 100.

Table 2.6.3.1 Substrate specificity of FUT8. ND, Not Detected. Conversions were monitored by HPLC- ELSD, average of three replicates were shown.

Glycan substrate Conversion (%) N04 5.26 N010 75.6 N020 49.8 N030 32.8 N000 91.3 N110 63.4 N210 ND N211 67.8 N212 69.4 N213 70.4 N214 40.1 N215 34.8 All other N- ND glycans

Figure 2.6.3 The 77 N-glycans used as acceptor for FUT8 substrate specificity study. The preparation and characterization of all substrates were published previously6. The ones in the red square were found to be suitable acceptors for FUT8.

2.6.4 General methods for glycan synthesis and purification

Reactions catalyzed by B4GALT1, Hpa1,3FT, PmST1m and Pd2,6ST were performed and monitored as we described previously26. Reactions catalyzed by FUT8 were performed in 100 mM

MES buffer (pH 7.0), containing 2 mM glycan acceptor, 4 mM GDP-Fuc donor, FastAP (10 U/mL) and 0.1 mg/mL FUT8. Reactions were allowed to proceed for 6 h to overnight at 37 °C until substrates were totally converted to products (monitored by HPLC-ELSD as described above). The reactions were then quenched by freezing in -80 °C for 30 min, followed with concentration by lyophilization. HPLC-A210nm was then used to purify target glycans using a semi-preparative amide column (130 Å, 5 µm, 10 mm × 250 mm). The running conditions are solvent A: 100 mM ammonium formate (for glycans with Neu5Ac residues) or water (for glycans without Neu5Ac residues), pH 3.4; solvent B: acetonitrile; flow rate: 4 mL/min; B%: 70-55% within 25 min.

Products containing portions were then concentrated and lyophilized for characterization. The purity of each glycan was confirmed by HPLC-ELSD using an analytical Waters amide column

(130 Å, 5 µm, 4.6 mm × 250 mm). The running conditions are solvent A: 100 mM ammonium formate, pH 3.4; solvent B: acetonitrile; flow rate: 1 mL/min; B%: 65-50% within 25 min.

The molecular weight of each N-glycans was confirmed by MALDI-MS performed on

UltrafleXtreme MALDI TOF/TOF Mass Spectrometer (Bruker). Scan range of MS1 was according to the molecular weight of N-glycans, and reflector mode was used for N-glycan analysis. Mass spectra were obtained in both positive and negative extraction mode with the following voltage settings: ion source 1 (19.0 kV), ion source 2 (15.9 kV), and lens (9.3 kV). The reflector voltage was set to 20 kV. The laser was pulsed at 7 Hz and the pulsed ion extraction time was set to 400 ns. The laser power was kept in the 25–40% range. Structures of key intermediates and asymmetric

N-glycans with core-fucosylation were confirmed by 1H NMR.

2.6.5 HPLC profiles and MS data of purified N-glycans

N6110 (6.9 mg)

MALDI-MS, calculated: 1588.5761; found [M+Na]+: 1611.5692, [M+K]+: 1627.1403

4 x10 1611.5692 Intens. [a.u.] Intens. 1.5

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6111a (3.8 mg)

MALDI-MS, calculated: 1750.6290; found [M+Na]+: 1773.6207, [M+K]+: 1789.2301

4 x10 1773.6207 2.0 Intens. [a.u.] Intens.

1.5

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6111 (3.5 mg)

HPLC-ELSD, TR = 14.01 min

Dataf ile Name:N6111.lcd mV ELSD

1.25

1.00

0.75

0.50

0.25

0.00

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1624.5973; found [M+Na]+: 1647.5894

4 x10 1647.5894

2.0 Intens. [a.u.] Intens.

1.5

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6112 (1.0 mg)

HPLC-ELSD, TR = 15.45 min

Dataf ile Name:N6112.lcd mV ELSD 0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1915.6927; found [M-H]-: 1914.6880

4 x10 1914.6880

0.8 Intens. [a.u.] Intens.

0.6

0.4

0.2

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6113 (1.5 mg)

HPLC-ELSD, TR = 16.73 min

Dataf ile Name:N6113.lcd mV ELSD 3.0

2.5

2.0

1.5

1.0

0.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1915.6927; found [M-H]-: 1914.6896

1914.6896

8000 Intens. [a.u.] Intens.

6000

4000

2000

0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6122 (0.8 mg)

HPLC-ELSD, TR = 17.36 min

Dataf ile Name:N6122.lcd mV ELSD

1.50

1.25

1.00

0.75

0.50

0.25

0.00

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2077.7455; found [M-H]-: 2076.7399

4 x10 2076.7399 3.0

Intens. [a.u.] Intens. 2.5

2.0

1.5

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6123 (1.2 mg)

HPLC-ELSD, TR = 18.36 min

Dataf ile Name:N6123.lcd mV ELSD 2.5

2.0

1.5

1.0

0.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2077.7455; found [M-H]-: 2076.7392

4 x10 2076.7392

Intens. [a.u.] Intens. 2.0

1.5

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6144 (0.8 mg)

HPLC-ELSD, TR = 20.12 min

Dataf ile Name:N6144.lcd mV 0.8 AD2

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2223.8034; found [M-H]-: 2222.7993

2222.7993

400 Intens. [a.u.] Intens.

300

200

100

0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N211a (4.6 mg)

MALDI-MS, calculated: 1604.5710; found [M+Na]+: 1627.5650

4 x10 1627.5650 1.50

Intens. [a.u.] Intens. 1.25

1.00

0.75

0.50

0.25

0.00 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N211 (4.4 mg)

MALDI-MS, calculated: 1478.5393; found [M+Na]+: 1501.5305, [M+K]+: 1517.5030

4 x10 1501.5305 1.2

Intens. [a.u.] Intens. 1.0

0.8

0.6

0.4

0.2

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6211 (4.0 mg)

HPLC-ELSD, TR = 13.75 min

Dataf ile Name:N6211.lcd mV 4.5 ELSD

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1624.5973; found [M+Na]+: 1647.5885, [M+K]+: 1663.5652

4 x10 1647.5885

6 Intens. [a.u.] Intens.

0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6212 (1.3 mg)

HPLC-ELSD, TR = 15.19 min

Dataf ile Name:N6212.lcd mV ELSD

0.4

0.3

0.2

0.1

0.0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1915.6927; found [M-H]-: 1914.6891

4 x10 1914.6891

1.25 Intens. [a.u.] Intens.

1.00

0.75

0.50

0.25

0.00 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6213 (1.8 mg)

HPLC-ELSD, TR = 16.51 min Dataf ile Name:N6213.lcd mV ELSD 3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1915.6927; found [M-H]-: 1914.6897

4 x10 1914.6897 1.25 Intens. [a.u.] Intens. 1.00

0.75

0.50

0.25

0.00 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6222 (0.9 mg)

HPLC-ELSD, TR = 17.32 min

Dataf ile Name:N6222.lcd mV 2.25 ELSD

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2077.7455; found [M-H]-: 2076.7422

4 x10 2076.7422

1.25 Intens. [a.u.] Intens.

1.00

0.75

0.50

0.25

0.00 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6223 (1.3 mg)

HPLC-ELSD, TR = 18.39 min

Dataf ile Name:N6223.lcd mV ELSD 3.0

2.5

2.0

1.5

1.0

0.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2077.7455; found [M-H]-: 2076.7418

2076.7418

3000 Intens. [a.u.] Intens.

2000

1000

0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

N6244 (0.9 mg)

HPLC-ELSD, TR = 20.11 min

Dataf ile Name:N6244 2.lc d mV 0.7 AD2

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 2223.8034; found [M-H]-: 2222.7986

4 x10 2222.7986

1.5 Intens. [a.u.] Intens.

1.0

0.5

0.0 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 m/z

2.6.6 ESI-MS and 1H NMR data of purified N604

N604 (0.4 mg)

ESI-MS, calculated: 894.3329; found [M+H]+: 895.3384, [M+Na]+: 917.3202

1H NMR of N604

1 H NMR (D2O, 500 MHz): d 6.18 (s, 1 H), 6.17 (s, 1 H),4.65 (s, 1 H), 4.41 (brs, 2 H), 4.20-

4.30 (m, 5 H), 4.08-4.12 (m, 2 H), 4.56-4.00 (m, 20 H), 2.12 (s, 3 H, NHAc), 2.07 (s, 3 H, NHAc),

1.25 (t, J = 5.4 Hz, 3 H, Fuc-CH3)

2.6.7 NMR spectra and data of purified core-fucosylated N-glycans

1H NMR of N6122

1 H NMR (D2O, 500 MHz): d 5.17 (d, J = 2.9 Hz, 0.5 H, GlcNAc-1 H1 of a isomer), 5.11

(s, 1 H, Man2 H-1), 4.91 (s, 1 H, Man3 H-1), 4.88 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of a isomer),

4.87 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of b isomer), 4.75-4.80 (overlap with D2O, 1 H, Manb H-1),

4.65-4.68 (m, 1.5 H, GlcNAc-1 H-1 of b isomer, GlcNAc-2 H-1), 4.57 (d, J = 7.9 Hz, 1 H, GlcNAc-

3 H-1), 4.56 (d, 1 H, J = 6.6 Hz, 1 H, GlcNAc-4 H-1), 4.53 (d, J = 7.9 Hz, 1 H, Gal-1 H-1), 4.46

(d, 1 H, J = 7.8 Hz, 1 H, Gal-2 H-1), 4.24 (brs, 1 H), 4.18 (d, J = 1.9 Hz, 1 H), 4.08-4.11 (m, 3 H),

3.46-4.00 (m, 60 H), 2.75 (dd, J = 12.5, 4.6 Hz, 1 H, Neu5Ac H-3eq), 2.08 (s, 3 H, Ac), 2.04 (s, 6

H, Ac), 2.03 (s, 3 H, Ac),2.02 (s, 3 H, Ac), 1.79 (t, J = 12.5 Hz, 1 H, Neu5Ac H-3ax), 1.21 (d, J =

6.4 Hz, 1.5 H, Fuc H-6), 1.20 (d, J = 6.4 Hz, 1.5 H, Fuc H-6)

1H NMR of N6123

1 H NMR (D2O, 500 MHz): d 5.17 (d, J = 2.9 Hz, 0.5 H, GlcNAc-1 H-1 of a isomer), 5.12

(s, 1 H, Man2 H-1), 4.92 (s, 1 H, Man3 H-1), 4.88 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of a isomer),

4.87 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of b isomer), 4.75-4.85 (overlap with D2O, 1 H, Manb H-1),

4.65-4.69 (m, 1.5 H, GlcNAc-1 H-1 of b isomer, GlcNAc-2 H-1), 4.59 (d, J = 7.9 Hz, 1 H, GlcNAc-

3 H-1), 4.57 (d, J = 7.8 Hz, 1 H, GlcNAc-4 H-1), 4.46 (d, 1 H, J = 7.8 Hz, 1 H, Gal-1 H-1), 4.43

(d, 1 H, J = 7.9 Hz, 1 H, Gal-2 H-1), 4.24 (brs, 1 H), 4.18 (d, J = 1.8 Hz, 1 H), 4.07-4.09 (m, 2 H),

3.46-4.00 (m, 61 H), 2.66 (dd, J = 12.5, 4.7 Hz, 1 H, Neu5Ac H-3eq), 2.08 (s, 3 H, Ac), 2.06 (s, 3

H, Ac), 2.04 (s, 3 H, Ac),2.03 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 1.71 (t, J = 12.5 Hz, 1 H, Neu5Ac H-

3ax), 1.21 (d, J = 6.3 Hz, 1.5 H, Fuc H-6), 1.20 (d, J = 6.3 Hz, 1.5 H, Fuc H-6)

1H NMR of N6144

1 H NMR (D2O, 500 MHz): d 5.16 (d, J = 2.8 Hz, 0.5 H, GlcNAc-1 H1 of a isomer), 5.10

(d, J = 3.9 Hz, 1 H, Fuc-2 H-1), 5.09 (s, 1 H, Man2 H-1), 4.92 (s, 1 H, Man3 H-1), 4.87 (d, J = 3.7

Hz, 0.5 H, Fuc H-1 of a isomer), 4.86 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of b isomer), 4.75-4.83

(overlap with D2O, 1 H, Manb H-1), 4.65-4.68 (m, 1.5 H, GlcNAc-1 H-1 of b isomer, GlcNAc-2

H-1), 4.56-4.58 (m, 2 H, GlcNAc-3 H-1, GlcNAc-4 H-1), 4.41-4.43 (m, 2 H, Gal-1 H-1, Gal-2 H-

1), 4.23 (brs, 1 H), 4.17 (d, J = 1.9 Hz, 1 H), 4.07-4.12 (m, 3 H), 3.42-3.99 (m, 64 H), 2.65 (dd, J

= 12.5, 4.7 Hz, 1 H, Neu5Ac H-3eq), 2.08 (s, 3 H, Ac), 2.05 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.01

(s, 3 H, Ac), 2.00 (s, 3 H), 1.70 (t, J = 12.5 Hz, 1 H, Neu5Ac H-3ax), 1.21 (d, J = 6.3 Hz, 1.5 H,

Fuc H-6), 1.20 (d, J = 6.3 Hz, 1.5 H, Fuc H-6), 1.15 (d, J = 6.5 Hz, 2 H, Fuc H-6), 1.11 (d, J = 6.5

Hz, 1 H, Fuc H-6).

1H NMR of N6222

1 H NMR (D2O, 500 MHz): d 5.19 (d, J = 2.6 Hz, 0.5 H, GlcNAc-1 H-1 of a isomer), 5.13

(s, 1 H, Man2 H-1), 4.93 (s, 1 H, Man3 H-1), 4.90 (d, J = 3.5 Hz, 0.5 H, Fuc H-1 of a isomer),

4.89 (d, J = 3.5 Hz, 0.5 H, Fuc H-1 of b isomer), 4.75-4.80 (overlap with D2O, 1 H, Manb H-1),

4.70 (d, J = 8.0 Hz, 0.5 H, GlcNAc-1 H-1 of b isomer), 4.67 (d, J = 7.7 Hz, 1 H, GlcNAc-2 H-1),

4.55-4.59 (m, 3 H, GlcNAc-3 H-1, GlcNAc-4 H-1, Gal-1 H-1), 4.47 (d, 1 H, J = 7.8 Hz, 1 H, Gal-

2 H-1), 4.26 (brs, 1 H), 4.20 (brs, 1 H), 4.10-4.14 (m, 3 H), 3.46-4.00 (m, 60 H), 2.77 (dd, J = 12.4,

4.4 Hz, 1 H, Neu5Ac H-3eq), 2.11 (s, 3 H, Ac), 2.06 (s, 3 H, Ac), 2.05 (s, 6 H, Ac),2.04 (s, 3 H,

Ac), 1.81 (t, J = 12.4 Hz, 1 H, Neu5Ac H-3ax), 1.22 (d, J = 6.0 Hz, 1.5 H, Fuc H-6), 1.21 (d, J =

6.0 Hz, 1.5 H, Fuc H-6)

1H NMR of N6223

1 H NMR (D2O, 500 MHz): d 5.14 (d, J = 2.9 Hz, 0.5 H, GlcNAc-1 H-1 of a isomer), 5.10

(s, 1 H, Man2 H-1), 4.92 (s, 1 H, Man3 H-1), 4.88 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of a isomer),

4.87 (d, J = 3.7 Hz, 0.5 H, Fuc H-1 of b isomer), 4.75-4.85 (overlap with D2O, 1 H, Manb H-1),

4.64-4.68 (m, 1.5 H, GlcNAc-1 H-1 of b isomer, GlcNAc-2 H-1), 4.55-4.58 (m, 2 H, GlcNAc-3

H-1, GlcNAc-4 H-1), 4.42-4.45 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.23 (s, 1 H), 4.17 (brs, 1 H),

4.07-4.12 (m, 2 H), 3.46-4.00 (m, 61 H), 2.65 (dd, J = 12.5, 4.7 Hz, 1 H, Neu5Ac H-3eq), 2.08 (s,

3 H, Ac), 2.04 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.70 (t, J = 12.5

Hz, 1 H, Neu5Ac H-3ax), 1.20 (d, J = 6.4 Hz, 1.5 H, Fuc H-6), 1.19 (d, J = 6.4 Hz, 1.5 H, Fuc H-

1H NMR of N6244

1 H NMR (D2O, 500 MHz): d 5.16 (d, J = 2.9 Hz, 0.6 H, GlcNAc-1 H-1 of a isomer), 5.11

(s, 1 H, Man2 H-1), 5.10 (d, J = 3.8 Hz, 1 H, Fuc-2 H-1), 4.89 (s, 1 H, Man3 H-1), 4.87 (d, J = 3.8

Hz, 0.4 H, Fuc H-1 of a isomer), 4.86 (d, J = 3.8 Hz, 0.6 H, Fuc H-1 of b isomer), 4.75-4.83

(overlap with D2O, 1 H, Manb H-1), 4.64-4.68 (m, 1.4 H, GlcNAc-1 H-1 of b isomer, GlcNAc-2

H-1), 4.56-4.59 (m, 2 H, GlcNAc-3 H-1, GlcNAc-4 H-1), 4.42-4.44 (m, 2 H, Gal-1 H-1, Gal-2 H-

1), 4.24 (brs, 1 H), 4.17 (d, J = 2.4 Hz, 1 H), 4.06-4.09 (m, 3 H), 3.46-4.00 (m, 64 H), 2.65 (dd, J

= 12.3, 4.5 Hz, 1 H, Neu5Ac H-3eq), 2.08 (s, 3 H, Ac), 2.05 (s, 3 H, Ac), 2.02 (s, 6 H, Ac), 2.01

(s, 3 H, Ac), 1.70 (t, J = 12.3 Hz, 1 H, Neu5Ac H-3ax), 1.20 (d, J = 6.3 Hz, 1.2 H, Fuc H-6), 1.20

(d, J = 6.3 Hz, 1.8 H, Fuc H-6), 1.15 (d, J = 6.6 Hz, 3 H, Fuc H-6).

2.7 References

1. A. Varki and J. B. Lowe, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 2. Y. Y. Zhao, M. Takahashi, J. G. Gu, E. Miyoshi, A. Matsumoto, S. Kitazume and N. Taniguchi, Cancer science, 2008, 99, 1304-1310. 3. R. A. Dwek, Chemical reviews, 1996, 96, 683-720. 4. B. Imperiali and S. E. O'Connor, Current opinion in chemical biology, 1999, 3, 643-649. 5. A. Helenius and M. Aebi, Science, 2001, 291, 2364-2369. 6. A. Helenius and M. Aebi, Annual review of biochemistry, 2004, 73, 1019-1049. 7. R. L. Shields, J. Lai, R. Keck, L. Y. O'Connell, K. Hong, Y. G. Meng, S. H. Weikert and L. G. Presta, J Biol Chem, 2002, 277, 26733-26740. 8. T. Shinkawa, K. Nakamura, N. Yamane, E. Shoji-Hosaka, Y. Kanda, M. Sakurada, K. Uchida, H. Anazawa, M. Satoh, M. Yamasaki, N. Hanai and K. Shitara, J Biol Chem, 2003, 278, 3466-3473. 9. A. Okazaki, E. Shoji-Hosaka, K. Nakamura, M. Wakitani, K. Uchida, S. Kakita, K. Tsumoto, I. Kumagai and K. Shitara, Journal of molecular biology, 2004, 336, 1239- 1249. 10. W. Li, R. Yu, B. Ma, Y. Yang, X. Jiao, Y. Liu, H. Cao, W. Dong, L. Liu, K. Ma, T. Fukuda, Q. Liu, T. Ma, Z. Wang, J. Gu, J. Zhang and N. Taniguchi, Journal of immunology, 2015, 194, 2596-2606. 11. N. Taniguchi and Y. Kizuka, Advances in cancer research, 2015, 126, 11-51. 12. S. S. Pinho and C. A. Reis, Nature reviews. Cancer, 2015, 15, 540-555. 13. Y. Aoyagi, O. Isokawa, T. Suda, M. Watanabe, Y. Suzuki and H. Asakura, Cancer, 1998, 83, 2076-2082. 14. Y. Aoyagi, Y. Suzuki, M. Isemura, M. Nomoto, C. Sekine, K. Igarashi and F. Ichida, Cancer, 1988, 61, 769-774. 15. K. Taketa, Y. Endo, C. Sekiya, K. Tanikawa, T. Koji, H. Taga, S. Satomura, S. Matsuura, T. Kawai and H. Hirai, Cancer research, 1993, 53, 5419-5423. 16. E. Miyoshi, K. Noda, Y. Yamaguchi, S. Inoue, Y. Ikeda, W. Wang, J. H. Ko, N. Uozumi, W. Li and N. Taniguchi, Biochim Biophys Acta, 1999, 1473, 9-20. 17. F. Geng, B. Z. Shi, Y. F. Yuan and X. Z. Wu, Cell research, 2004, 14, 423-433. 18. R. Saldova, W. B. Struwe, K. Wynne, G. Elia, M. J. Duffy and P. M. Rudd, International journal of molecular sciences, 2013, 14, 15636-15654. 19. X. Wang, S. Inoue, J. Gu, E. Miyoshi, K. Noda, W. Li, Y. Mizuno-Horikawa, M. Nakano, M. Asahi, M. Takahashi, N. Uozumi, S. Ihara, S. H. Lee, Y. Ikeda, Y. Yamaguchi, Y. Aze, Y. Tomiyama, J. Fujii, K. Suzuki, A. Kondo, S. D. Shapiro, C. Lopez-Otin, T. Kuwaki, M. Okabe, K. Honke and N. Taniguchi, Proc Natl Acad Sci U S A, 2005, 102, 15791-15796. 20. J. R. Wilson, D. Williams and H. Schachter, Biochem Biophys Res Commun, 1976, 72, 909-916. 21. G. D. Longmore and H. Schachter, Carbohydr Res, 1982, 100, 365-392. 22. J. A. Voynow, R. S. Kaiser, T. F. Scanlin and M. C. Glick, J Biol Chem, 1991, 266, 21572-21577. 23. J. Kaminska, M. C. Glick and J. Koscielak, Glycoconjugate journal, 1998, 15, 783-788.

24. K. Paschinger, E. Staudacher, U. Stemmer, G. Fabini and I. B. Wilson, Glycobiology, 2005, 15, 463-474. 25. B. Echeverria, J. Etxebarria, N. Ruiz, A. Hernandez, J. Calvo, M. Haberger, D. Reusch and N. C. Reichardt, Analytical chemistry, 2015, 87, 11460-11467. 26. L. Li, Y. Liu, C. Ma, J. Qu, A. D. Calderon, B. Wu, N. Wei, X. Wang, Y. Guo, Z. Xiao, J. Song, G. Sugiarto, Y. Li, H. Yu, X. Chen and P. G. Wang, Chemical science, 2015, 6, 5652-5661. 27. I. Prahl and C. Unverzagt, Tetrahedron Letters, 2000, 41, 10189-10193. 28. S. Eller, R. Schuberth, G. Gundel, J. Seifert and C. Unverzagt, Angewandte Chemie, 2007, 46, 4173-4175. 29. D. Ott, J. Seifert, I. Prahl, M. Niemietz, J. Hoffman, J. Guder, M. Mönnich and C. Unverzagt, European Journal of Organic Chemistry, 2012, 2012, 5054-5068. 30. B. Sun, B. Srinivasan and X. Huang, Chemistry, 2008, 14, 7072-7081. 31. S. Serna, S. Yan, M. Martin-Lomas, I. B. Wilson and N. C. Reichardt, Journal of the American Chemical Society, 2011, 133, 16495-16502. 32. S. Q. Fan, W. Huang and L. X. Wang, J Biol Chem, 2012, 287, 11272-11281. 33. W. Huang, J. Giddens, S. Q. Fan, C. Toonstra and L. X. Wang, Journal of the American Chemical Society, 2012, 134, 12308-12318. 34. D. J. Wasilko, S. E. Lee, K. J. Stutzman-Engwall, B. A. Reitz, T. L. Emmons, K. J. Mathis, M. J. Bienkowski, A. G. Tomasselli and H. D. Fischer, Protein expression and purification, 2009, 65, 122-132. 35. K. Martin, R. Talukder, F. C. Hay and J. S. Axford, The Journal of rheumatology, 2001, 28, 1531-1536. 36. C. R. O'Riordan, A. L. Lachapelle, J. Marshall, E. A. Higgins and S. H. Cheng, Glycobiology, 2000, 10, 1225-1233. 37. E. M. Kratz, M. Ferens-Sieczkowska, R. Faundez and I. Katnik-Prastowska, Glycoconjugate journal, 2014, 31, 51-60. 38. N. Kui Wong, R. L. Easton, M. Panico, M. Sutton-Smith, J. C. Morrison, F. A. Lattanzio, H. R. Morris, G. F. Clark, A. Dell and M. S. Patankar, J Biol Chem, 2003, 278, 28619- 28634. 39. G. Sugiarto, K. Lau, Y. Li, Z. Khedri, H. Yu, D. T. Le and X. Chen, Molecular bioSystems, 2011, 7, 3021-3027. 40. H. Yu, S. Huang, H. Chokhawala, M. Sun, H. Zheng and X. Chen, Angewandte Chemie, 2006, 45, 3938-3944. 41. S. W. Lin, T. M. Yuan, J. R. Li and C. H. Lin, Biochemistry, 2006, 45, 8108-8116. 42. H. Yu, H. Yu, R. Karpel and X. Chen, Bioorganic & medicinal chemistry, 2004, 12, 6427-6435. 43. M. M. Muthana, J. Qu, Y. Li, L. Zhang, H. Yu, L. Ding, H. Malekan and X. Chen, Chemical communications, 2012, 48, 2728-2730. 44. G. Zhao, W. Guan, L. Cai and P. G. Wang, Nature protocols, 2010, 5, 636-646. 45. H. Ihara, Y. Ikeda and N. Taniguchi, Glycobiology, 2006, 16, 333-342.

3 FUT8: FROM BIOCHEMISTRY TO SYNTHESIS OF CORE-FUCOSYLATED N-

GLYCANS.

The work presented in this chapter is based on an accepted invited review on Pure and

Applied Chemistry. This chapter includes work by Angie D. Calderon, Lei Li and Peng G. Wang.

3.1 Abstract

Glycosylation is a major post-translational modification of proteins. Modification in structure of N-glycans leads to many diseases. One of such modifications is core a-1,6 fucosylation, which is only found in eukaryotes. For this reason, lots of research has been done on approaches to synthesize a1,6-core-fucosylated N-glycans both chemically and enzymatically, in order to have well-defined structures that can be used as probes for glycan analysis and identifying functions of glycan-binding proteins. This review will focus on FUT8, the enzyme responsible for core fucosylation in mammals and the strategies that have been developed for the synthesis of a1,6 core-fucosylated N-glycans so far.

Keywords: FUT8, fucosyltransferases, specificity studies, core fucosylation, alpha-1,6- fucosyltransferase.

3.2 Introduction

Post-translational modifications are very important since they influence folding, stability, signaling, transport and life span of proteins.1, 2 One major post-translational modification is N- glycosylation, the alteration which is usually involved in biological processes such as: cell adhesion, infections, immunogenicity, and tumor metastasis.3-6 N-glycans are also proven to influence protein biosynthesis, folding, molecular stability, solubility, and serum half-life.5, 7-9 The study of human glycosyltransferases (hGTs) is important to the understanding of these important cellular event as well as their involvement in major diseases.2, 6, 10-14 N-glycan structures in nature can be very complex and diverse due to the great degree of modification that takes place. a1,6 core-fucosylation is a major N-glycan modification in eukaryotic systems.

This review will focus on the biochemical properties of human a1,6 fucosyltransferase

(FUT8) and its biosynthetic applications. FUT8 is the enzyme responsible for all a1,6 core fucosylation in mammals. This enzyme catalyzes the transfer of an L-fucose residue from GDP- b-L-fucose (GDP-Fuc) to the inner GlcNAc of asparagine linked complex glycopeptides to fucosylate with an a1,6-linkage (Figure 3.2.1). 15-20

Figure 3.2.1 Sample reaction catalyzed by a1,6 fucosyltransferase (FUT8).

FUT8 belongs to the fucosyltransferase family (EC 2.4.1.68) and it is encoded in humans by the FUT8 gene. FUT8 is a type II membrane protein on the Golgi apparatus.15 Human FUT8 has 575 aa without a consensus N-glycosylation and its localized on chromosome 14. FUT8 is structurally and genetically different from other a1,2, a1,3, and a1,4 fucosyltransferases.13, 21

The 3D structure of human FUT8 has been solved using recombinant proteins expressed by baculovirus infected Sf21 cells in 2007, which revealed a similar catalytic region to that of GT-B glycosyltransferases.22

Core-fucosylation modification of N-glycans is found and conserved in vertebrates and invertebrates such as Caenorhabditis elegans and Drosophila melanogaster.19, 23-25 In contrast to the mammalian FUT8, C. elegans and D. melanogaster FUT8 has one non-conserved potential

N-glycosylation site. 19 Many groups have studied the % identity of different animal species of

FUT8 and they are listed in Table 2.6.7.1.

Table 2.6.7.1 % identity of FUT8 between animal species.26, 27 Animal species Size (aa) % Identity H. sapiens 575 100 M. musculus 575 96 B. taurus 575 95 S. scrofa 575 93 G. gallus 232 88 D. melanogaster 619 60 C. elegans 541 36

FUT8 has great biological significance, loss of core-fucosylation modification can lead to growth retardation, emphysema, Alzheimer, and even death. 20, 28 In addition, core-fucosylation is related to the regulation and function of many glycoproteins. One example is the lack of N- glycan core-fucosylation in human IgG1 leads to enhanced antibody-dependent cellular cytotoxicity (ADCC).23, 29-31 Core-fucosylation also regulates the functions of immunoglobulin, for example, in IgG B cell receptors, it is required for the antigen recognition in humoral immune response.32, 33 Numerous studies have shown the overexpression of FUT8 contributes to the development, progression, malignancy, recurrence, invasive and metastatic properties of cancer

65 cells.10, 12-14, 34 Modifications in core-fucosylation have been linked to human diseases including liver cancer, pancreatic cancer, lung cancer, breast cancer, etc.35, 36 Furthermore, a1,6 core fucosylation has great potential as a cancer biomarker as shown by a-fetoprotein, which is highly core-fucosylated in hepatocellular carcinoma, but not other liver diseases.12, 37-40 Other core- fucosylated glycoproteins such as E-cadherin and CA125 were also found to be potential cancer biomarkers depending on their core fucosylation level.41, 42 Core a1,6-fucosylation is a major modification and there is strong evidence for its importance in biological events.

3.3 FUT8 specificity studies

Due to its importance and medical relevance many research groups have studied the substrate specificity for this enzyme. 17, 23, 43, 44 Some research groups have expressed soluble forms of C. elegans and D. melanogaster FUT8 in Pichia pastoris. They had also showed that invertebrate’s FUT8 has the same biochemical function as vertebrate FUT8 and recognizes biantennary N-glycans as substrates.19

It has been widely elucidated regarding the substrate specificity of FUT8 in vivo and in vitro. Human FUT8 is active in the absence of metal cations and was shown to be inhibited by

Ni+2, Cu+2, and Zn+2. Optimal buffers were cacodylate and Mes when compared to Tris-HCl and sodium phosphate buffers at pH 7.0.18 A library of 77 well defined N-glycans synthesized by our group composed of three types of unlabeled N-glycans structure types: oligomannose, complex and hybrid as well as four simple glycan structures was used to reveal details of FUT8 substrate specificity. The results showed that recombinant human FUT8 expressed by our group recognized 11 out of the 77 substrates assayed.45 Our results support the previous substrate specificity studies that suggest that GlcNAc at the a-3 mannose branch of the substrate is required for the catalytic activity of the enzyme. 16-19, 24, 43, 45-48 Human and C. elegans FUT8

66 have relax substrate preference towards the a1,6-mannose branch, since it fucosylated asymmetric N-glycans.45, 48, 49 Human FUT8 recognition towards asymmetric N-glycans with an a1,3-linked fucose to the terminal N-acetyllactosamine (LacNAc) or sialyl LacNAc structures on the a1,6-mannose branch is less efficient.45 N-glycan without an a1,3-mannose branch was still recognized by human FUT8 with a neglectable percent conversion.45 Bisecting N-glycans and galactosylated GlcNAc residue at a1,3-mannose branch prevent FUT8’s activity.16, 17, 45

Reichardt’s group, determined that C. elegans FUT8 can also core fucosylated xylose containing bi-antennary N-glycans relevant in invertebrates and plants.50 In general, FUT8 showed to have a strict requirement towards the a1,3-mannose branch, and very relaxed requirement towards the a1,6-mannose branch. 16-19, 24, 43, 45-48

FUT8 substrate recognition in vivo varies slightly according to findings by various groups. Even though the presence of GlcNAc at the a3 mannose branch catalyzed by N- acetylglucosaminyltransferase I (Gn-T I) is considered to be a prerequisite for core fucosylation by FUT8, there are traces of high mannose core fucosylated glycans in vivo.51, 52 a1,6 Core- fucosylated high mannose structures were identified from lysosomal proteins such as rat liver alkaline phosphatase51, porcine cathepsin D,52 human glucuronidase,53 and bovine mannosidase.54 Therefore, these structures were thought to be generated by N- acetylglucosaminidase hydrolysis of core fucosylated hybrid type N-glycans. After various groups detected the presence of a1,6 core-fucosylated high mannose structures from natural proteins expressed in Gn-T I knockout CHO55, and HEK293S56 cell lines, the presence of a Gn-T

I independent fucosylation pathway was confirmed.

Wang’s group was able to demonstrate that FUT8 is the sole enzyme responsible for a1,6 core-fucosylation in the Gn-T I independent fucosylation pathway. They also suggested the

67 level of core-fucosylation depends on the nature of the recombinant proteins. This was demonstrated by the expression of erythropoietin (EPO), GM-CSF and ectodomain of FcgIIIA receptor recombinant proteins in HEK293S Gn-T I-/- cell line with an a1,6 core-fucosylation percentage of 50%, 30%, and 3 % respectively. They confirmed core-fucosylation was the sole activity of FUT8 by the expression of EPO in stable HEK293 Gn-T I-/- cell line with both knockdown or overexpressed FUT8. Their results showed knockdown cell line produced pure

Man5GlcNAc2 glycoform, compared to EPO from FUT8 overexpressing cell line, which produced completely core fucosylated Man5GlcNAc.57

3.4 Traditional versus chemo-enzymatic synthesis of core fucosylated N-glycans.

Synthesis of core-fucosylated N-glycans has been targeted from a variety of aspects.

Since 1996 Unverzagt’s group synthesized the first a1,6 core-fucosylated N-glycan structure synthetically.58 They synthesized an a1,6 core-fucosylated bi-antennary octasaccharide58 and nonasaccharide,59 as well as an a1,6 core-fucosylation bisecting penta-antennary dodecasaccharide, with an azide group for the generation of glycopeptides.60 The a1,6 core- fucose was introduced at the last step of the chemical synthesis. For the a-fucosylation a p- methoxybenzyl (MPM) substituted thiofucoside was used so that it could be selectively removed

61 by oxidation. The thiofucoside was activated by Bu4NBr/CuBr2 for coupling to the desired oligosaccharide.58-60, 62 They were also able to optimize the synthesis of a biantennary core- fucosylated N-glycan by using a key building block selectively functionalized trisaccharide that allows glycosylation in a modular way including fucosylation at any stage of the synthesis.63

Huang’s group has also successfully synthesized an a-2,3 sialylated core-fucosylated biantennary N-glycan dodecasaccharide by combining three modules/blocks in one-pot.64

In the more recent years’ different approaches have been developed to generate more complex core fucosylated N-glycan structures in more efficient ways. As described by Yu’s review on thioglycosides, there are many advantages and disadvantages to using thioglycosides

(such as thiofucoside) for the chemical synthesis of these complex glycan structures.65 Some of the more known advantages are: 1. activation under extremely mild conditions, 2. stability under wide range of conditions, and 3. easily accessible. However, waste resulting from these compounds and their promoters have very unpleasant smell and are toxic. The requirement for promoters also leads to the generation of many side reactions. These major disadvantages are a real problem to the environment and human health, therefore not a favorable route for industrial applications.65

Several groups have been able to merge chemical synthesis with enzymatic synthesis in order to develop more efficient synthetic ways of producing large quantities of core-fucosylated

N-glycans. Wang’s group was one of the pioneers in chemically synthesize an a1,6 core- fucosylated N-linked disaccharide to test the specificity of several endo-b-N- acetylglucosaminidases such as Endo A, Endo M, Endo D, etc. Endoglycosidases from

Flavobacterium meningosepticum Endo-F2 and Endo-F3 were the only ones that recognized

Fuca1,6GlcNAc-N-Fmoc/peptide substrate. The substrate and donor were chemically synthesized. The substrate was synthesized to have an a1,6-fucosylation. Using Endo-F2 and

Endo-F3 they were able to synthesize both sialylated and asialylated a1,6 core-fucosylated N- glycans. With this approach they generate a full sized CD52 glycopeptide antigen both with sialic acid and a1,6 core-fucose (Figure 3.4.1).66

Figure 3.4.1 Chemo-enzymatic synthesis of core-fucosylated glycopeptide and glycoprotein using Endo- F3 and mutated Endo-F3 D165A to transfer biantennary and triantennary complex N-glycans.66, 67

Wang’s group years later demonstrated that by mutating Endo-F3 to Endo-F3 D165A it is possible to transfer not only sialylated and asialylated bi-antennary N-glycans oxazolines, but also tri-antennary N-glycan oxazolines to form a complex tri-antennary a1,6 core-fucosylated glycopeptide or glycoprotein. The donor tri-antennary oxazoline was semi-synthesized by digestion of fetal bovine fetuin using Endo-F3 followed by purification and desialylation using sialidase. Conversion into tri-antennary glycan oxazoline was done in a single step using excess of 2-chloro-1,3- dimethylimidazolinium chloride and triethylamine in water. This new endoglycosidase based glycosynthase can transfer bi-antennary and tri-antennary complex N- glycans to glycopeptides and glycoproteins such as Rituximab and porcine fibrinogen (Figure

3.4.1). Making it possible to remodel therapeutic antibody glycosylation pattern including those with a1,6 core-fucosylation.67

Reichardt, used an 18 glycan array to test the specificity of Caenorhabditis elegans a1,6 fucosyltransferase (CeFUT8) expressed in Pichia pastoris. The specificity of the enzyme toward the different glycan structure was assayed by four lectins: Lens culinaris agglutinin (LCA),

Aleuria aurantia lectin (AAL), Pisum sativum agglutinin (PSA), Aspergillus oryzae lectin

(AOL), and anti-horseradish peroxidase antibody (anti-HRP). AAL was the only lectin that bound with good selectivity and sensitivity to all a1,6 core-fucosylated structures. CeFUT8 recognized A8-A17 (10 out 18 substrates assayed) (Figure 3.4.2A). After specificity assays for the CeFUT8 the findings were applied to synthesize two a1,6 core-fucosylated N-glycans in solution. A9 and A14 were used as substrates for the synthesis of a1,6 core-fucosylated N- glycans in solution. The final product Compound 5 and 7 were confirmed by NMR and MS

(Figure 3.4.2C, Figure 3.4.2D). All the substrates applied for the specificity studies and scale up synthesis for core a-1,6 fucosylation were chemically synthesized. 47

Figure 3.4.2 A) CeFUT8 specificity assay using structures on glycan array. B) All substrates were chemically synthesized with linker. C&D) Compound 5 and 7 were synthesized using CeFUT8 from A9 and A14 respectively, in solution and the structure was confirmed by NMR and MS.47

Reichardt’s group also determined that N-glycan structures containing xylose can be a1,6 core-fucosylated by CeFUT8. Using 3 chemically synthesized N-glycan structures containing xylose (G3, G5, G6), 5 glycosyltransferases (α1,6- fucosyltransferase (CeFUT8), α1,3- fucosyltransferase (AtFucTA/CeFUT1), β1,4-galactosyltransferase (GalT), β1,4-N-acetyl-

71 galactosyltransferase (GalNAcT), LeX-type α1,6-fucosyltransferase (CeFUT6)), and 1 N-acetyl- glucosaminidase they were able to synthesize by chemo-enzymatic extension 14 a1,6 core- fucosylated invertebrate and plant N-glycans containing xylose Figure 3.4.3.50

Figure 3.4.3 Scheme of chemo-enzymatic synthesis of 14 a1,6 core fucosylated invertebrate and plant N- glycans containing xylose.50 Reaction Conditions: a. CeFUT8, b. GalT, c. AtFucTA/CeFUT1, d. GalNAcT, e. CeFUT6, f. N-acetyl-glucosaminidase.

Reichardt’s group also used a similar chemo-enzymatic strategy, reviewed before, to generate a library of 16 13C-labeled N-glycans useful as internal standards for absolute quantification by MALDI-TOF. 8 out of the 16 N-glycans synthesized by their group were core- fucosylated N-glycans and only 3 were asymmetrical. The chemically synthesized core structure

(Compound 2) that’s partially protected and isotope labeled (Figure 3.4.4) was used for the extension using 3 enzymes (β-1,4-GalT, β-1,4-N-acetyl-glucosaminidase, α-2,3-SialylT) to generate the N-glycan library. In order to generate the 8 core-fucosylated N-glycans Compound

2 was deprotected by hydrogenation before a1,6 fucosylation.49

Figure 3.4.4 Compound 2 before deprotection of R1=Bn by hydrogenation. In red, incorporated 13C isotopes. Scheme of chemo-enzymatic synthesis of CF-glycans.6, 49 Reaction Conditions: a. b-1,4-GalT, b. 10% Pd/C, H2 c. a1,6 FucT d. N-acetyl-glucosaminidase, e. a-2,3-SialylT.

Our group has also contributed to the preparation of N-glycans using a chemo-enzymatic strategy. 72 N-glycans were chemo-enzymatically synthesized following extension starting from

8 core chemically synthesized N-glycans to generate 62 isomers and 4 core fucosylated glycans using recombinant human a-1,6 fucosyltransferase (FUT8). The core fucosylated structures generate (N6030, N6000, N6211, N6212) were synthesized from N030, N000, N211, and N212

(Figure 3.4.5). N030 and N000 were chemically synthesized as part of the core structures used for extension. N211 and N212 were chemo-enzymatically synthesized.48 The a1,6 core- fucosylated N-glycans generated were the same as previously reported by Reichardt’s group.

Figure 3.4.5 CF-glycans generated by Wang lab.48

A complete study on the specificity of recombinant human FUT8 was done by our group using the 77 N-glycan library that was mentioned above. Using the specificity of the enzyme a chemo-enzymatic strategy was developed for the synthesis of a panel of a1,6 core-fucosylated asymmetric N-glycans. The generation of 14 core-fucosylated N-glycans was achieved by following the scheme in Figure 3.4.6.45

The scheme used two chemically synthesized starting N-glycans N110 and N210 as substrates. For the enzymatic extension of asymmetric core-fucosylated N-glycans N110 and

N210 had a protected GlcNAc at the a6 or a3 mannose branch, respectively. All the core- fucosylated N-glycans were confirmed by MALDI-MS and HPLC retention time. Core fucosylated isomers N6122, N6123, N6244, N6222, N6223, and N6144 were confirmed by 1H-

NMR as well.45

Figure 3.4.6 (A) Scheme for chemo-enzymatic synthesis of asymmetric a1,6 core-fucosylated N-glycans starting with N110 as a substrate. B) Scheme for chemo-enzymatic synthesis of asymmetric a1,6 core fucosylated N-glycans starting with N210 as a substrate.45 Reaction conditions: a. FUT8, GDP-Fuc, 2+ FastAP; b. b4GALT1, UDP-Gal, FastAP, Mn ; c. 30% ammonium hydroxide : H2O (1 : 10), 6 h; d) PmST1m, CMP-Neu5Ac; e. Pd2,6ST, CMP-Neu5Ac; f. Hpa1,3FT, GDP-Fuc, FastAP, Mn2+. FUT8, human a1,6-fucosyltransferase; b4GALT1, bovine b1,4-galactosyltransferase; PmST1m, Pasteurella multocida a2,3-sialyltransferase 1 mutant E271F/R313Y; Pd2,6ST, Photobacterium damselae a 2,6- sialyltransferase; Hpa1,3FT, c-terminal 66 amino acids truncated Helicobacter pylori a1,3- fucosyltransferase; FastAP, thermo-sensitive alkaline phosphatase.

3.5 Conclusion

In conclusion, FUT8 the only mammalian enzyme responsible for core a1,6 fucosylation, has been widely studied by many research groups. The crystal structure has been determined as well as its metal, buffer and pH preferences. The substrate specificity for the enzyme was studied in detailed and the results suggest there is a strict requirement towards the a1,3-mannose branch, but very relax requirements towards the a1,6-mannose branch. Many ways to synthesize core a1,6 fucosylated N-glycans have been explored both chemically and enzymatically. These structures are valuable as standards for glycan analysis, and glycan-binding studies. New approaches to chemo-enzymatically synthesize complex a1,6 core-fucosylated N-glycans have allowed for the rapid accessibility to well-defined structures. Future focused arrays containing core-fucosylated N-glycans will allow for identifying prospective antigens for vaccine design.

These new chemo-enzymatic strategies will play a crucial role in the synthesis of potential antigenic a-1,6 fucosylated N-glycans for glycoconjugate vaccines.47

3.6 Acknowledgements

This work was supported by the National Institutes of Health (U01GM0116263 to P. G.

Wang and L. Li), and Graduate Assistance in Areas of National Need (GAANN P200A150308).

3.7 References

1. R. D. Cummings and J. D. Esko, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 2. M. Kuhlmann and A. Covic, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2006, 21 Suppl 5, v4-8. 3. A. Varki and J. B. Lowe, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 4. Y. Y. Zhao, M. Takahashi, J. G. Gu, E. Miyoshi, A. Matsumoto, S. Kitazume and N. Taniguchi, Cancer science, 2008, 99, 1304-1310. 5. R. A. Dwek, Chemical reviews, 1996, 96, 683-720. 6. A. M. Sinclair and S. Elliott, Journal of pharmaceutical sciences, 2005, 94, 1626-1635. 7. B. Imperiali and S. E. O'Connor, Current opinion in chemical biology, 1999, 3, 643-649. 8. A. Helenius and M. Aebi, Science, 2001, 291, 2364-2369. 9. A. Helenius and M. Aebi, Annual review of biochemistry, 2004, 73, 1019-1049. 10. Y. Ito, A. Miyauchi, H. Yoshida, T. Uruno, K. Nakano, Y. Takamura, A. Miya, K. Kobayashi, T. Yokozawa, F. Matsuzuka, N. Taniguchi, N. Matsuura, K. Kuma and E. Miyoshi, Cancer Lett, 2003, 200, 167-172. 11. K. Kamio, T. Yoshida, C. Gao, T. Ishii, F. Ota, T. Motegi, S. Kobayashi, R. Fujinawa, K. Ohtsubo, S. Kitazume, T. Angata, A. Azuma, A. Gemma, M. Nishimura, T. Betsuyaku, K. Kida and N. Taniguchi, Biochem Biophys Res Commun, 2012, 424, 112-117. 12. E. Miyoshi, K. Moriwaki, N. Terao, C. C. Tan, M. Terao, T. Nakagawa, H. Matsumoto, S. Shinzaki and Y. Kamada, Biomolecules, 2012, 2, 34-45. 13. Y. Yamaguchi, J. Fujii, S. Inoue, N. Uozumi, S. Yanagidani, Y. Ikeda, M. Egashira, O. Miyoshi, N. Niikawa and N. Taniguchi, Cytogenet Cell Genet, 1999, 84, 58-60. 14. X. Wang, J. Chen, Q. K. Li, S. B. Peskoe, B. Zhang, C. Choi, E. A. Platz and H. Zhang, Glycobiology, 2014, 24, 935-944. 15. J. R. Wilson, D. Williams and H. Schachter, Biochem Biophys Res Commun, 1976, 72, 909-916. 16. G. D. Longmore and H. Schachter, Carbohydr Res, 1982, 100, 365-392. 17. J. A. Voynow, R. S. Kaiser, T. F. Scanlin and M. C. Glick, J Biol Chem, 1991, 266, 21572-21577.

18. J. Kaminska, M. C. Glick and J. Koscielak, Glycoconjugate journal, 1998, 15, 783-788. 19. K. Paschinger, E. Staudacher, U. Stemmer, G. Fabini and I. B. Wilson, Glycobiology, 2005, 15, 463-474. 20. X. Wang, S. Inoue, J. Gu, E. Miyoshi, K. Noda, W. Li, Y. Mizuno-Horikawa, M. Nakano, M. Asahi, M. Takahashi, N. Uozumi, S. Ihara, S. H. Lee, Y. Ikeda, Y. Yamaguchi, Y. Aze, Y. Tomiyama, J. Fujii, K. Suzuki, A. Kondo, S. D. Shapiro, C. Lopez-Otin, T. Kuwaki, M. Okabe, K. Honke and N. Taniguchi, Proc Natl Acad Sci U S A, 2005, 102, 15791-15796. 21. Y. Yamaguchi, Y. Ikeda, T. Takahashi, H. Ihara, T. Tanaka, C. Sasho, N. Uozumi, S. Yanagidani, S. Inoue, J. Fujii and N. Taniguchi, Glycobiology, 2000, 10, 637-643. 22. H. Ihara, Y. Ikeda, S. Toma, X. Wang, T. Suzuki, J. Gu, E. Miyoshi, T. Tsukihara, K. Honke, A. Matsumoto, A. Nakagawa and N. Taniguchi, Glycobiology, 2007, 17, 455- 466. 23. H. Ihara, Y. Ikeda and N. Taniguchi, Glycobiology, 2006, 16, 333-342. 24. M. P. Kotzler, S. Blank, F. I. Bantleon, E. Spillner and B. Meyer, Biochim Biophys Acta, 2012, 1820, 1915-1925. 25. M. Costache, P. A. Apoil, A. Cailleau, A. Elmgren, G. Larson, S. Henry, A. Blancher, D. Iordachescu, R. Oriol and R. Mollicone, J Biol Chem, 1997, 272, 29721-29728. 26. P. Coullin, R. P. Crooijmans, M. A. Groenen, R. Heilig, R. Mollicone, R. Oriol and J. J. Candelier, Cytogenet Genome Res, 2002, 97, 234-238. 27. C. Javaud, F. Dupuy, A. Maftah, J. C. Michalski, R. Oriol, J. M. Petit and R. Julien, Mol Biol Evol, 2000, 17, 1661-1672. 28. K. Akasaka-Manya, H. Manya, Y. Sakurai, B. S. Wojczyk, S. L. Spitalnik and T. Endo, Glycoconjugate journal, 2008, 25, 775-786. 29. H. Imai-Nishiya, K. Mori, M. Inoue, M. Wakitani, S. Iida, K. Shitara and M. Satoh, BMC Biotechnol, 2007, 7, 84. 30. R. L. Shields, J. Lai, R. Keck, L. Y. O'Connell, K. Hong, Y. G. Meng, S. H. Weikert and L. G. Presta, J Biol Chem, 2002, 277, 26733-26740. 31. T. Shinkawa, K. Nakamura, N. Yamane, E. Shoji-Hosaka, Y. Kanda, M. Sakurada, K. Uchida, H. Anazawa, M. Satoh, M. Yamasaki, N. Hanai and K. Shitara, J Biol Chem, 2003, 278, 3466-3473. 32. A. Okazaki, E. Shoji-Hosaka, K. Nakamura, M. Wakitani, K. Uchida, S. Kakita, K. Tsumoto, I. Kumagai and K. Shitara, Journal of molecular biology, 2004, 336, 1239- 1249. 33. W. Li, R. Yu, B. Ma, Y. Yang, X. Jiao, Y. Liu, H. Cao, W. Dong, L. Liu, K. Ma, T. Fukuda, Q. Liu, T. Ma, Z. Wang, J. Gu, J. Zhang and N. Taniguchi, Journal of immunology, 2015, 194, 2596-2606. 34. L. Muinelo-Romay, S. Villar-Portela, E. Cuevas Alvarez, E. Gil-Martin and A. Fernandez-Briera, Human pathology, 2011, 42, 1740-1750. 35. N. Taniguchi and Y. Kizuka, Advances in cancer research, 2015, 126, 11-51. 36. S. S. Pinho and C. A. Reis, Nature reviews. Cancer, 2015, 15, 540-555. 37. Y. Aoyagi, O. Isokawa, T. Suda, M. Watanabe, Y. Suzuki and H. Asakura, Cancer, 1998, 83, 2076-2082. 38. Y. Aoyagi, Y. Suzuki, M. Isemura, M. Nomoto, C. Sekine, K. Igarashi and F. Ichida, Cancer, 1988, 61, 769-774.

39. K. Taketa, Y. Endo, C. Sekiya, K. Tanikawa, T. Koji, H. Taga, S. Satomura, S. Matsuura, T. Kawai and H. Hirai, Cancer research, 1993, 53, 5419-5423. 40. E. Miyoshi, K. Noda, Y. Yamaguchi, S. Inoue, Y. Ikeda, W. Wang, J. H. Ko, N. Uozumi, W. Li and N. Taniguchi, Biochim Biophys Acta, 1999, 1473, 9-20. 41. F. Geng, B. Z. Shi, Y. F. Yuan and X. Z. Wu, Cell research, 2004, 14, 423-433. 42. R. Saldova, W. B. Struwe, K. Wynne, G. Elia, M. J. Duffy and P. M. Rudd, International journal of molecular sciences, 2013, 14, 15636-15654. 43. M. C. Shao, C. W. Sokolik and F. Wold, Carbohydr Res, 1994, 251, 163-173. 44. E. Staudacher and L. Marz, Glycoconjugate journal, 1998, 15, 355-360. 45. A. D. Calderon, Y. Liu, X. Li, X. Wang, X. Chen, L. Li and P. G. Wang, Org Biomol Chem, 2016, 14, 4027-4031. 46. M. P. Kotzler, S. Blank, F. I. Bantleon, M. Wienke, E. Spillner and B. Meyer, ACS chemical biology, 2013, 8, 1830-1840. 47. S. Serna, S. Yan, M. Martin-Lomas, I. B. Wilson and N. C. Reichardt, Journal of the American Chemical Society, 2011, 133, 16495-16502. 48. L. Li, Y. Liu, C. Ma, J. Qu, A. D. Calderon, B. Wu, N. Wei, X. Wang, Y. Guo, Z. Xiao, J. Song, G. Sugiarto, Y. Li, H. Yu, X. Chen and P. G. Wang, Chemical science, 2015, 6, 5652-5661. 49. B. Echeverria, J. Etxebarria, N. Ruiz, A. Hernandez, J. Calvo, M. Haberger, D. Reusch and N. C. Reichardt, Analytical chemistry, 2015, 87, 11460-11467. 50. K. Brzezicka, B. Echeverria, S. Serna, A. van Diepen, C. H. Hokke and N. C. Reichardt, ACS Chem Biol, 2015, 10, 1290-1302. 51. T. Endo, T. Fujiwara, Y. Ikehara and A. Kobata, Eur J Biochem, 1996, 236, 579-590. 52. T. Takahashi, P. G. Schmidt and J. Tang, J Biol Chem, 1983, 258, 2819-2830. 53. D. R. Howard, M. Natowicz and J. U. Baenziger, J Biol Chem, 1982, 257, 10861-10868. 54. V. Faid, G. Evjen, O. K. Tollersrud, J. C. Michalski and W. Morelle, Glycobiology, 2006, 16, 440-461. 55. A. I. Lin, G. A. Philipsberg and R. S. Haltiwanger, Glycobiology, 1994, 4, 895-901. 56. M. Crispin, D. J. Harvey, V. T. Chang, C. Yu, A. R. Aricescu, E. Y. Jones, S. J. Davis, R. A. Dwek and P. M. Rudd, Glycobiology, 2006, 16, 748-756. 57. Q. Yang and L. X. Wang, J Biol Chem, 2016, 291, 11064-11071. 58. J. Seifert and C. Unverzagt, Tetrahedron Letters, 1996, 37, 6527-6530. 59. I. Prahl and C. Unverzagt, Tetrahedron Letters, 2000, 41, 10189-10193. 60. S. Eller, R. Schuberth, G. Gundel, J. Seifert and C. Unverzagt, Angewandte Chemie, 2007, 46, 4173-4175. 61. T. M. Slaghek, Y. Nakahara, T. Ogawa, J. P. Kamerling and J. F. G. Vliegenthart, Carbohydrate Research, 1994, 255, 61-85. 62. S. Sato, M. Mori, Y. Ito and t. Ogawa, Carbohydrate Research, 1986, 155, C6-C10. 63. D. Ott, J. Seifert, I. Prahl, M. Niemietz, J. Hoffman, J. Guder, M. Mönnich and C. Unverzagt, European Journal of Organic Chemistry, 2012, 2012, 5054-5068. 64. B. Sun, B. Srinivasan and X. Huang, Chemistry, 2008, 14, 7072-7081. 65. G. Lian, X. Zhang and B. Yu, Carbohydrate Research, 2015, 403, 13-22. 66. W. Huang, J. Li and L. X. Wang, Chembiochem, 2011, 12, 932-941. 67. J. P. Giddens, J. V. Lomino, M. N. Amin and L. X. Wang, J Biol Chem, 2016, 291, 9356- 9370.

4 ENZYMATIC SEPARATION OF ASYMMETRIC BI- AND TRI-ANTENNARY N-

GLYCAN ISOMERS USING FUT8 AND GN-TV

The work presented in this chapter is based on a manuscript in preparation for submission to

Chemical Communication. This chapter includes compounds synthesized by Angie D. Calderon,

Lei Li, NMR data by Jun Zhou and LacZ expressed by Wanyi Guan, and Zhigang Wu.

4.1 Abstract

Human Gn-T V substrate specificity was studied using 17 structurally-defined N-glycans as acceptors. The enzyme showed a strict requirement towards the a1,6-mannose branch, and very relax requirement towards the a1,3-mannose branch. A strategy for the efficient enzymatic separation of asymmetric N-glycan isomers was developed taking advantage of the strict substrate specificity of recombinant human Gn-T V and previously reported recombinant human FUT8 glycosyltransferases.

Keywords: FUT8, Gn-T V, glycosyltransferases, Spodoptera frugiperda (Sf9), fucosylation.

4.2 Introduction

Glycosylation plays an important role in nature. Asparagine-linked glycosylation (N- glycosylation) is one of the most important post-translational modifications of eukaryotic proteins. N-glycans have been shown to modulate protein’s structure and function in many ways.

N-glycan structures were found to be very important in a variety of biological processes, including cell adhesion, immune responses and tumor metastasis.1-3 On the other hand, N- glycans can influence proteins folding, stability, and immunogenicity, among others.3-6 The natural complexity in the biosynthesis of N-glycans have led to the generation of very diverse and complex glycan structures. Synthetic and chemo-enzymatic ways for the generation of well- defined complex symmetric and asymmetric glycan structures as standards have been widely

79 studied by many groups including ours. 7-13 The generation of a large library of well-defined asymmetric N-glycan isomers was recently reported by our group.14 Current methods for the separation and characterization of N-glycan isomer structures, have been focused on the analysis of small quantities for analytical purposes. Methods such as ESI-MS/MS, IMS-MS/MS, nano-

LC-MS, HILIC-UPLC, and CE-LIF-MS among others, have successfully accomplished the separation and characterization of complex N-glycan isomer structures.15-24 Isolation and separation of isomers from natural, enzymatic or synthetic sources in large scale have proven to be challenging with current methods and technology. For this reason, a simple and efficient strategy for the separation and production of large number of asymmetric N-glycan isomers is highly desirable. Human glycosyltransferases are well known for their very strict substrate specificities. Their specificity can be useful in the separation of N-glycan isomers. Previously, we and others reported on FUT8 strict a6-mannose branch preference.25

Human acetylglucosaminyltransferases V (Gn-T V) is responsible for the transfer of N- acetylglucosamine (GlcNAc) from a uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to the natural acceptor to generate a b1,6GlcNAc tri-antennary N-glycan structures.26 Based on several studies on association of L-PHA lectin it was indicated that Gn-T V and its b1,6GlcNAc branching on N-glycans is implicated in advance cancers, tumor progression, and poor prognosis of epithelial origin cancers such as breast, and gastric cancers among others.27-42 The overexpression of this enzyme was linked to the regulation of cell surface glycoprotein receptors such as EGF, TGFb, and T cell receptors among others.43-46 A small peptide sequence in Gn-T V also showed to stimulate angiogenesis effects.47 Gn-T V knockout mouse appeared normal at birth, but as adults they displayed aberrant phenotypes such as T cell hypersensitivity, and autoimmune sensitivity among others. In all of the models Gn-T V deletion was traced back to

80 altered PKB and ERK signaling pathways.43, 48-51 Gn-T V is regulated by ras-raf-ets2 signaling pathway activated by over expression of oncogenes such as her2-neu observed in 20-30% of human breast cancers.52-55 From previous studies it is known that the enzyme does not recognize the bisected N-glycan product from Gn-T III.56-61 This enzyme has also previously shown some preference towards the a1,3-mannose branch terminal GlcNAc residue.26, 62-64

In this study, recombinant human Gn-T V was overexpressed using TIPs methodology65, purified and its detailed substrate specificity was studied against 17 well-defined N-glycans

(Figure 4.2.1). A comprehensive study was necessary to confirm such susbtste specificity, and structurally well-defined substrate N-glycans previously synthesized by our group represent perfect candidates for testing.14 An efficient strategy for the enzymatic separation of asymmetric

N-glycan isomers was developed (See Figure 4.6.6 and Figure 4.6.7).

This strategy is useful for the generation of asymmetric bi-, tri- and tetra-antennary complex

N-glycan isomer structures. The strategy takes advantage of Gn-T V and previously reported FUT8 high substrate specificity. N-linked bi- and tri-antennary core glycans were successfully isolated from egg yok and fetuin, respectively.66-68 Possible N-glycan isomers were generated by digestion of bi- and tri-antennary core glycans using commercial PNGase F, Neuraminidase and b- galactosidase from E.coli k12 (Ec-LacZ).

Gn-T V catalyzed reaction β6 β2 100 a6 b4 b4 a6 b4 b4 b2 b2 β2 β2 N000 a3 β2 a3 N7000 90 a3 a6 b2 b2 b4 b4 β6 a3 a6 80 β2 b4 a6 a6 b4 b4 a6 b4 b4 β2 b4 b4 β4 β4 N3000 N3700 70 b2 a3 a3 a3 b2 b2 b2 b2 β2 β2 a3 a6 a6 a3 a3 a6 b4 b4 60 b4 b4 b4 b4 b4 b2 b2 b4 b4 a3 b2 b2 b2 a3 a6 4 β2 β4 β2 50 a3 a6 β2 β b4 a3 a6 b4 a3 a6 b4 b4 a3 a6 b4 b4 b4 b4 40 b4 b4 a3 β2 β4 β2

Conversion (%) b4 b2 b2 b4 b4 a3 a3 a6 b4 b4 b4 b4 a6 30 b2 b2 b2 b4 a3 b4 b2 b2 β2 β4 a3 a3 a6 b4 b4 20 b4 b4 a3 a6 a3 a6 b4 b4 b4 b4 b4 b4 10

0 N6000 N000 N020 N040 N050 N210 N110 N111 N112 N113 N114 N115 N3001 N3000 N3111 N3361 Symbols: GlcNAc Man Gal L-fuc Peracetylation

Figure 4.2.1 Gn-T V catalyzed reaction and substrate specificity of human Gn-T V using 17 N-glycans as potential acceptors,(See Table 4.6.8.1 and Table 4.6.9.1 for conversion percentage towards each glycan). The average conversion percentage of three replicates for each acceptor was shown.

4.3 Results and Discussion

Large scale separation and characterization of N-glycan isomers has proven to be challenging in the past, thus complicating the generation of asymmetric N-glycans in large scale.

Herein, we present a novel strategy for the generation of asymmetric N-glycans completely from natural sources such as fetuin and egg yolk. FUT8 and Gn-T V substrate specificity was applied to the enzymatic separation of asymmetric bi-and tri-antennary N-glycans. FUT8 substrate specificity was studied in detailed previously.25 Gn-T V was expressed, purified and its detailed substrate specificity was assayed in order to be used in the synthetic scheme.

4.3.1 Generation of asymmetric biantennary and triantennary N-glycans

A. Generation of possible bi-antennary asymmetric N-glycan isomers β4 β2 a6 b4 b4

α6 β4 β2 α6 β4 β2 β4 β2 β2 β2 a6 a6 a3 a6 a6 b4 b4 PNGase F b4 b4 α-neuraminidase b4 b4 LacZ N211 b4 b4 2 β4 β2 α6 β4 β2 β4 β β2 β2 α6 a3 a3 a3 a3 N000 N003 N001 a6 b4 b4

β4 β2 a3 N111

B. Generation of possible tri-antennary asymmetric N-glycan isomers N3001 N3001 -1 Galactose residue -2 Galactose residues β2 β4 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 a3 N3111 a3 N3161 2 β4 β β4 β2 β4 β2 4 2 β4 β2 β β β4 β2 β2 a6 b4 b4 a6 a6 b4 a6 b4 b4 b4 a6 b4 b4 b4 b4 β4 β4 PNGase F β4 4 β4 LacZ β β4 β4 a3 α-neuraminidase a3 N3001 a3 N3211 a3 N3261 a3 N3000 β2 β2 β4 β4 β2 β4 β2 β2

β4 β2 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 β4 a3 N3311 a3 N3361 β2 β2

Figure 4.3.1 A. Scheme for the synthesis of possible asymmetric N-glycan isomers from SGP from isolated from egg yolk. B. Scheme for the synthesis of possible asymmetric N-glycan isomers from fetal bovine serum. N-glycan boxed in red are the identified asymmetric N-glycan generated from lacZ digestion.

b-galactosidase (LacZ) expressed from E. coli K12 was applied for the generation of asymmetric N-glycan isomers from purified substrates N001 and N3001 (Figure 4.3.1). Bi- antennary substrate N001 and tri-antennary N3001 were isolated from the digestion of sialylglycopeptide (SGP) from egg yolk66 and fetuin from fetal bovine serum67-71, respectively, using commercially available PNGase F and Neuraminidase (See pg. 92 §4.6.3 for a more detail description of the isolation and digestion of N3001). N3001 was well characterized by HPLC-

ELSD, MALDI-MS (See pg.103 §4.6.11), and 1H-NMR, 13C NMR, and multiple 2D-NMR experiments (HSQC, HMBC, and HSQC-TOCSY) (See pg.112 §4.6.12).

The substrate specificity of Ec-LacZ towards the bi-antennary and tri-antennary N- glycans has previously suggested b-galactosidase from E.coli to cleave the galactose residue at the a6 mannose branch slower and yielding an equimolar isomer mixture.72, 73 For this reason,

83 first we monitored the digestion reaction of N001 with LacZ every hour for 8 hours and overnight to determine the best time to collect the intermediate product with only 1 galactose

(N111/N211) residue for characterization (Figure 4.6.4). N3001 was also recognized by LacZ as a substrate for galactose digestion as shown by HPLC profile pre-purification. (Figure 4.6.5).

After prolonged incubation of acceptor with LacZ, a mixture of products was generated (N3001,

II, III, and N3000). N3001 without 1 galactose residue (Compound II) was the major digestion product. Each compound was purified and characterized by HPLC-ELSD (See pg.103 §4.6.11),

MALDI-MS (See pg.103 §4.6.11), and Compound II by 1H-NMR (See pg.112 §4.6.12).

4.3.2 Substrate Specificity for human Gn-T V

Human Gn-T V was chosen for substrate specificity study and synthetic purpose.

Expression was achieved via a Titerless Infected-cell Preservation and Scale-up (TIPS) approach65 using a baculovirus system (see http://glycoenzymes.ccrc.uga.edu for system and construction details) provided by Dr. Jarvis from the University of Wyoming. After one-step Ni-Sepharose

Excell (GE Healthcare) affinity purification, recombinant human Gn-T V was obtained in a scale of 3 mg/L of cultures as shown by western-blot (Figure 4.6.1). Substrate specificity study was then performed in reactions with a total volume of 10 µL containing an N-glycan acceptor (50

µM), donor UDP-GlcNAc (10 mM), MES buffer (100 mM, pH 7.0), MgCl2 (15 mM), 0.2% Triton

X-100, 1 mg/ml BSA, and 0.5 mg/mL of the recombinant human Gn-T V. After 1 h of incubation at 37 °C, the reactions were quenched by boiling, followed by analysis using mass spectrometry

(MS) and high performance liquid chromatography (HPLC) monitored by fluorescence detector

(RF-10A XL, Shimadzu). (See pg. 99 §4.6.8). Bi-antennary N-glycan acceptors and tri-antennary

N-glycans used for the specificity studies are listed in Table 4.6.8.1 and Table 4.6.7.1, respectively.

Gn-T V recognized N-glycans with an agalactosylated and unprotected GlcNAc residue on the a1,6-mannose branch (N000, N040, N050, and N6000), but not those either, without a GlcNAc residue (N020), galactosylated (N211) or with protected GlcNAc residue (N110) on the branch.

Among these N-glycans, Gn-T V showed the highest activity towards bi-antennary complex N- glycan structures terminated with GlcNAc at the non-reducing end of both a1,3- and a1,6- mannose branches N000 (72.3% conversion) and slightly decreased activities towards N6000

(61.2% conversion), N210 (45.5% conversion), and N050 (45.6% conversion) indicated that a1,6 fucosylation, protection or removal of GlcNAc residue at the a1,3-mannose branch can be well tolerated. Removal of a1,3-mannose branch N040 (24.0% conversion), galactosylation N111

(26.4% conversion), a1,6 sialyation N113 (37.5% conversion) and a1,3 fucosylation N114 (29.7% conversion) will further decrease the efficiency of Gn-T V. The presence of a2,3 sialic acid N112

(10.6% conversion) and N115 (ND), respectively is not preferred by Gn-T V. Gn-T V product from Sialyl Lewis x structure N115 was only detected after overnight reaction. Gn-T V also recognized tri-antennary N-glycans (N3000, N3111, and N3361) completely after prolonged time

(N3000, N3111 and N3361). The enzyme did not recognize N3001 (See Table 4.6.9.1) Overall, our substrate specificity study using a library of 17 structurally defined N-glycans confirmed that

Gn-T V has a strict requirement towards the a1,6-mannose branch, and a very relaxed preference towards the a1,3-mannose branch.

4.3.3 Enzymatic separation of N-glycan isomers

FUT8 and Gn-T V substrate specificity was used to determine the branch preference of

LacZ towards N001 and N3001. FUT8 was previously reported by our group and others to have a strict requirement towards b1,2-GlcNAc on the a1,3-mannose branch, but very relax towards

85 the a1,6-mannose branch.25 Here, we reported Gn-T V to have a strict requirement towards b1,2

GlcNAc on the a1,6-mannose branch, but very relax towards the a1,3-mannose branch.

β4 β2 a6 b4 b4 a6 A Asymmetric β2 glycan extension a3 N6211 B FUT8 N6211

β4 β2 a6 b4 b4 C β4 β2 β2 β2 a6 b4 b4 LacZ a3 N211 a6 b4 b4 N111 β4 β2 β2 a3 β2 a3 N001 a6 b4 N000 b4 D β4 β2 a3 N111 N6211

Gn-T V E β6 N111 β2 a6 b4 b4 Asymmetric β4 β2 glycan extension a3 N3001

Figure 4.3.2 A. LacZ 3 h digestion of N001 and identification of possible isomer intermediate using FUT8 and Gn-T V. Reactions were monitored by MALDI-MS. B. LacZ 3 h digestion intermediate incubated with FUT8 overnight. C. LacZ 3 h digestion intermediate incubated with Gn-T V overnight. D. Positive control N211 substrate incubated with FUT8 overnight. D. Negative control N111 substrate incubated with FUT8 overnight.

After overnight incubation with FUT8 and Gn-T V of 3 h LacZ digested N001 intermediate, only the fucosylated product was detected by MALDI-MS (Figure 4.3.2). This result suggested LacZ has a preference towards the a3-mannose branch. The results needed to be further investigated by performing a substrate specificity assay of LacZ against isomers N111-

AA and N211-AA (See Table 4.6.7.1). After overnight incubation with LacZ N111-AA was fully digested compared to N211-AA which was only 28.6% digested. From our specificity assay results LacZ demonstrated to have a preference towards N111-AA compared to N211-AA.

In order to confirm our results, 1H-NMR and HSQC experiments were performed on the purified

LacZ digested product after 3h in order to confirm the glycosidic linkage profile of the intermediate product. However, structural elucidation could not be achieved without HMBC and

HSQC-TOCSY spectra (See pg.112 §4.6.12). Alternatively, the structure was revealed as N211

86 bearing a Gal residue at the C-6 branch by comparison with previously reported spectrum14 and not an isomer mixture. From our results we can conclude, the digestion of N001 by LacZ will generate a mixture of N000 and N211. The condition for the reaction can be adjusted for the generation of mostly the asymmetric N211 product or N000.

N3001 -1 Galactose residue N3001 -2 Galactose residues β2 β4 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 a3 N3111 a3 N3161 2 β4 β β4 β2 4 2 β4 β2 β β β4 β2 β2 a6 b4 a6 b4 a6 b4 b4 b4 a6 b4 b4 b4 β4 4 β4 LacZ β β4 β4 a3 N3001 a3 N3211 a3 N3261 a3 N3000 2 β4 β2 β4 β β2 β2

β4 β2 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 β4 a3 N3311 a3 N3361 β2 β2

Figure 4.3.3 Scheme of possible isomer structures without 1 and 2 galactose residues after LacZ digestion.

Each of the purified isomer products from the LacZ digestion of N3001 (Figure 4.6.5) were applied as Gn-T V substrates (Table 4.6.9.1). All of the substrates with exception of

N3001 were GlcNAcylated by Gn-T V completely after prolonged incubation. Separation strategy using FUT8 and Gn-T V was applied as before to determine the possible isomer structures of N3001 without 1 and 2 galactose residues. N3001 without 1 galactose residue was not recognized by FUT8, but after overnight incubation with Gn-T V it was completely

GlcNAcylated (Figure 4.3.4). Suggesting that from the possible isomers generated after digestion (Figure 4.3.3) the product generated without 1 galactose residue is N3111. N3111 was synthesize in a larger scale (2.0 mg) and was well characterized by HPLC-ELSD, MALDI-MS

(See pg.103 §4.6.11), and 1H-NMR, and multiple 2D-NMR experiments (HSQC, HMBC, and

HSQC-TOCSY) (See pg.112 §4.6.12).

A. N3001 -1 Galactose residue

β6 β2 a6 a6 b4 b4 b4 b4 β2 Gn-T V β4 β4 FUT8 X β4 a3 β4 a3 N37111 N3111 β2 β4 β2 β4 β4 β2 a6 Gn-T V b4 b4 FUT8 X β4 X a3 N3211

β4 β2

β4 β2 β4 β2 a6 Gn-T V a6 b4 b4 FUT8 b4 b4 a6 X β4 β4 β4 β4 a3 N3311 a3 N36311 B. β2 β2 5000 N37111 2069.7421

Intens. [a.u.] Intens. 4000

3000

2000

1000

0 1000 1200 1400 1600 1800 2000 2200 2400 m/z

Figure 4.3.4 A. Scheme for identification of possible isomer intermediate using FUT8 and Gn-T V. B. MALDI-MS analysis of N3001 without 1 galactose residue after overnight incubation with Gn-T V. No a1,6 core-fucosylated product detected after overnight incuation with FUT8. Reaction conditions: a) FUT8, GDP-Fuc, FastAP; b) Gn-T V, UDP-GlcNAc, Mg2+; BSA, Tritron 100-X. Red box indicates the isomers identified from the MALDI-MS results (See pg.103 §4.6.11).

Possible isomer without 2 galactose residues was recognized by both FUT8 and Gn-T V after overnight incubation (Figure 4.3.5). Suggesting that from the possible isomers generated after digestion (Figure 4.3.3) the product generated without 2 galactose residues is N3361.

N3361 was confirmed by HPLC-ELSD and MALDI-MS (See pg. 103 §4.6.11),

N3001 -2 Galactose residue

β6 β4 β2 a6 b4 b4 a6 b4 β2 Gn-T V b4 β4 β4 FUT8 X a3 N37161 a3 N3161 β2 β2 β4 β4 β4 β2 β4 β2 a6 b4 b4 a6 a6 b4 b4 β4 Gn-T V β4 FUT8 X a3 N36261 a3 N3261 β2 β2

β6 β2 β2 a6 b4 b4 a6 b4 b4 a6 b4 b4 a6 β2 β4 β4 4 β4 Gn-T V β4 FUT8 β β4 a3 N37361 a3 N3361 a3 N36361 β2 β2 β2

N36361 1850.6662 800 N36361

Intens. [a.u.] Intens. 600

400

200

0 1000 1200 1400 1600 1800 2000 m/z 1907.6851 125 N37361

Intens. [a.u.] Intens. 100

0 1000 1200 1400 1600 1800 2000 2200 2400 m/z

Figure 4.3.5 A. Scheme for identification of possible isomer intermediate using FUT8 and Gn-T V. B. MALDI-MS analysis of N3001 without 2 galactose residue after overnight incubation with FUT8 (N36361) and Gn-T V (N37111-AEAB). Reaction conditions: a) FUT8, GDP-Fuc, FastAP; b) Gn-T V, UDP-GlcNAc, Mg2+; BSA, Tritron 100-X. Red box indicates the isomers identified from the MALDI-MS results (See pg.103 §4.6.11). From the specificity and separation results the following symmetric (N37000 and

N36000), and asymmetric (N37361, N37111, and N36361) core tetra-antennary and a1,6 core fucosylated tri-antennary structures were synthesized. N37111, N36361, N37000, N36000, and

N37361 were confirmed by MALDI-MS and HPLC-ELSD profile (See pg. 103 §4.6.11).

4.4 Conclusion

In conclusion, recombinant human Gn-T V was overexpressed, purified and its substrate specificity assayed against 17 well defined N-glycans. Results showed a strict requirement towards the a1,6-mannose branch, and a very relax requirement towards the a1,3-mannose branch. Enzymatic separation of bi- and tri-antennary asymmetric N-glycan isomers was successful by taking advantage of the substrate specificity requirements of two well-studied human glycosyltransferase FUT8 and Gn-T V substrate specificity. Large scale separation of asymmetric N-glycans from natural sources is highly desirable for the generation of well-defined asymmetric N-glycan structures valuable as standards and probes for glycomic analysis and elucidation of glycan-binding protein functions.

4.5 Acknowledgements

This work was supported by National Institutes of Health (U01GM0116263 to P.G. Wang and L. Li) and National Cancer Institute (Contract SBIR: HHSN261201500021C to Chemily,

LLC). We are grateful to Dr. Donald Jarvis from the University of Wyoming for kindly providing the Sf9 cells and FUT8 baculovirus stock (NIH/NIGMS (P41GM103390 to Donald Jarvis), and

Graduate Assistance in Areas of National Need (GAANN P200A150308).

4.6 Experimental

4.6.1 Materials and enzymes

Sialylglycopeptide (SGP), was isolated from natural source egg yolk as previously reported66 and tri-antennary N-glycan (N3003) was isolated from natural source Fetuin67-71 from fetal bovine serum purchased from Sigma-Aldrich. Thermosensitive Alkaline Phosphatase

(FastAP) was purchased from Thermo Scientific. PNGase F and neuraminidase were purchased

90 from Sigma Aldrich. b-galactosidase (LacZ) from E.coli K12, recombinant human a-1,6- mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase (Gn-T V), and recombinant human core a1,6-fucosyltransferase (FUT8)25 were expressed and purified. Enzymes were then desalted against 50 mM Tris-HCl, 100 mM NaCl, and 50% glycerol, and stored at -20 °C for long term use.

Sugar nucleotides uridine 5’-diphospho-GlcNAc (UDP-GlcNAc) and guanosine 5’-diphospho-L- fucose (GDP-Fuc)74 were prepared as described previously.

4.6.2 Expression and Purification

b-galactosidase (LacZ) was expressed in E. coli using normal expression conditions: 100

µg/mL ampicillin, 0.1 mM IPTG, 16 degree 20 hours. For purification, 20 mM PBS, pH 7.4,

100 mM NaCl, 10 mM imidazole for wash buffer and 500 mM imidazole for elution buffer.

Recombinant human a-1,6-mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

(Gn-T V), and recombinant human core a1,6-fucosyltransferase (FUT8) were expressed using

Sf9 insect cells as previously described.25, 65 The baculovirus stocks for the expression of each of the hGTs were kindly provided by Dr. Donald Jarvis group from University of Wyoming NIH grant number RR005351. Enzymes were secreted into the medium, collected after 72 hours, followed by purification.

The purification of the secreted protein (Gn-T V and FUT8) was done by diluting the medium 1:1 with equilibration buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl) and loading the expression medium to an ÄKTA pure fast protein liquid chromatography system (FPLC) from

GE healthcare life sciences with a 1 mL pre-equilibrated His-Trap Excel affinity column (GE

Healthcare)75. The column was rinsed using wash buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl,

50 mM imidazole), and eluted with 10 mL elution buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl,

250 mM imidazole). Each enzyme was desalted and concentrated using Amicon YM-30

91 ultrafiltration membrane (Millipore) against desalting buffer (50 mM Tris-HCl pH 7.5).75 After desalting SDS-PAGE and Western blot were performed. SDS-PAGE was performed using a

10% gel the protein was transferred to PVDF membrane for western blot using iBlot transfer machine. The membrane was blocked using block solution (5% non-fat milk in TBS buffer pH

7.5 containing 0.05% Tween). Primary antibody used was anti-His mouse antibody 1:1000 dilution in block solution, and HRP-conjugated rabbit anti mouse IgG 1:1000 dilution for the secondary antibody. The protein was visualized by chemoluminescence using ECL system.

Enzyme concentration was assayed using commercially available Pierce BCA protein assay kit

(Thermo Fisher).

Figure 4.6.1 Western Blot for Gn-T V.

Figure 4.6.2 SDS-PAGE for Ec-LacZ expression and purification M. Premixed Protein Marker (High) (44.3-200 kDa); 1. Cell lysate; 2. Supernatant; 3. Pellet; 4. Unbound protein; 5-6. Wash; 7. Elution; 8. Diluted elution protein.

4.6.3 Generation of starting materials N001 and N3001 from natural source

A. Bi-antennary N-glycan from egg yolk

α6 β4 β2 α6 β4 β2 β4 β2 a6 a6 b4 b4 PNGase F a6 b4 b4 α-neuraminidase b4 b4 α6 β4 β2 α6 β4 β2 β4 β2 a3 a3 N003 a3 N001

B. Tri-antennary N-glycan from Fetuin (fetal bovine serum)

β4 β2 β4 β2 β4 β2 a6 a6 a6 b4 b4 PNGase F b4 b4 α-neuraminidase b4 b4 β4 β4 β4 β4 β4 β4 a3 a3 N3003 a3 N3001 β2 β2 β2 β4 β4 β4

Figure 4.6.3 A) Scheme for the generation of N001 in one pot reaction. B) Scheme for the generation of N3001 in one pot reaction.

N001 was generated from the one pot enzymatic digestion of SGP with PNGase F and a- neuraminidase (Figure 4.6.3). SGP was isolated from egg yolk by the method previously described by our group.66 N3001 was generated from the one pot enzymatic digestion of 100 mg

Fetuin from fetal bovine serum (Figure 4.6.3). Digestion reactions by PNGaseF and

Neuraminidase were performed in 50 mM NH4HC03 buffer (pH 8.0), containing 10 mM glycan acceptor and PNGaseF. Reactions were allowed to proceed for overnight at 37 °C until substrates were totally converted to intermediate product (monitored by HPLC-ELSD as described previously).25 The reaction was quenched by boiling. Precipitated protein was removed by centrifugation and Neuraminidase was added to the reaction. Reactions were allowed to proceed overnight at 37 °C until substrates were totally converted to intermediate product (monitored by

HPLC-ELSD as described previously).25 The reaction was quenched by boiling. Precipitated protein was removed by centrifugation. Sample was concentrated by rotatory evaporation and loaded on a G25 gel filtration column for purification. Purification was monitored using TLC with a solvent system of isopropanol : ammonium hydroxide : water (7:3:2). Each collected fraction was analyzed by HPLC-A210nm then used to purify target glycans using a semi-

93 preparative amide column (130 Å, 5 µm, 10 mm × 250 mm). The running conditions were solvent A: 100 mM ammonium formate, pH 3.2; solvent B: acetonitrile; flow rate: 4 mL/min;

B%: 65-50% within 25 min. Product containing fractions were then concentrated and lyophilized for characterization. N3001 was collected and lyophilized. 10 mg were isolated and characterized by HPLC-ELSD, MALDI-MS, and 1H-NMR.

4.6.4 Generation of asymmetric structures using LacZ

The generation of asymmetric structures was accomplished by the digestion of N001

(Figure 4.6.4) and N3001 (Figure 4.6.5) by LacZ. Digestion reactions by LacZ were performed in 100 mM Tris-HCl buffer (pH 7.5), containing 2 mM glycan acceptor and 1 mg/mL LacZ. A reaction time course was performed for N001 to determine the best time to collect the intermediate with 1 galactose (Figure 4.6.4). Reaction was sample every hour for 8 hours and 1 overnight sample. The percent conversion for the time course was monitor by HPLC-ELSD as previously described. 25 Larger scale reaction (10 mg) reactions were allowed to proceed for 3 h for N001 and 3 days for N3001 at 37 °C until substrates were totally converted to intermediate product (monitored by HPLC-ELSD as described previously).25 The reactions were then quenched by freezing in -80 °C for 30 min, followed with concentration by lyophilization.

HPLC-A210nm was then used to purify target glycans using a semi-preparative amide column (130

Å, 5 µm, 10 mm × 250 mm). The running conditions are solvent A: 100 mM ammonium formate

, pH 3.2; solvent B: acetonitrile; flow rate: 4 mL/min; B%: 65-50% within 25 min. Product containing fraction were then concentrated and lyophilized for characterization. The purity of each glycan was confirmed by HPLC-ELSD using an analytical Waters amide column (130 Å, 5

µm, 4.6 mm × 250 mm). The running conditions are solvent A: 100 mM ammonium formate, pH

3.2, solvent B: acetonitrile; flow rate: 1 mL/min; B%: 65-50% within 25 min.

β4 β2 a6 b4 b4 uV β4 β2 β2 10000 a6 b4 β2 a6 b4 LacZ a3 N211 b4 b4 β4 β2 β2 9000 a3 N001 β2 a3 N000 a6 b4 b4

β4 β2 8000 a3 N111

7000

6000 A B

5000 C 0 1 4000 2 3 3000 85% 4 5 2000 6 7 1000 8 9

0 53% 47% 10

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

Figure 4.6.4 LacZ digestion of N001 over time, reaction monitored by HPLC-ELSD. A) N001 control. B) N211 control. C) N000 control. 0) Negative control no enzyme. 1)Reaction 0 min. 2) Reaction 1 h. 3) Reaction 2 h. 4) Reaction 3 h, 85% conversion to intermediate product. 5) Reaction 4 h. 6) Reaction 5 h. 7) Reaction 6 h. 8) Reaction 7 h. 9) Reaction 8 h. 10) Reaction ov, 47% conversion to intermediate product, and 53% conversion to N000 product.

II III

N3001 -1 Galactose residue N3001 -2 Galactose residues β2 β4 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 a3 N3111 a3 N3161 IV 2 I β4 β β4 β2 4 2 β4 β2 β β β4 β2 β2 a6 b4 a6 b4 a6 b4 b4 b4 a6 b4 b4 b4 β4 4 β4 LacZ β β4 β4 a3 N3001 a3 N3211 a3 N3261 a3 N3000 2 β4 β2 β4 β β2 β2

β4 β2 β2 a6 b4 b4 a6 b4 b4 β4 β4 β4 β4 a3 N3311 a3 N3361 β2 β2 uV 500000

400000 II

300000 III I 200000 IV

100000

-100000

-200000

-300000

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n Figure 4.6.5 LacZ digestion of N3001 over 4 days, each intermediate product was purified and confirmed by MALDI-MS. I) N3001 II) Intermediate product without 1 galactose residue (N3111). III) Intermediate product without 2 galactose residues (N3361). IV) Completely digested product (N000).

4.6.5 Separation of N-glycan isomers using FUT8 and Gn-T V

The purified isomer intermediates were used as substrates for FUT8 and Gn-T V reactions. The reaction condition for FUT8 was: N-glycan substrate (5 mM), GDP-fucose (10 mM), MES buffer (100 mM, pH 7.0), excess FastAP), and excess FUT8 (0.5 mg/mL). The reaction condition for Gn-T V was: N-glycan substrate (5 mM), UDP-GlcNAc (10 mM), MES buffer (100 mM, pH 7.0), MgCl2 (15 mM), 0.2% Triton X-100, BSA (1 mg/ml), and excess Gn-

T V (0.5 mg/mL). Reactions were incubated at 37 °C overnight. The reactions were then quenched by boiling for 5 min. After centrifugation (13,000 g for 10 min), the supernatant was

96 analyzed by MALDI-MS. N001 digestion and enzymatic separation scheme is shown in Figure

4.6.6. N3001 digestion and enzymatic separation scheme is shown in Figure 4.6.7.

β4 β2 a6 b4 b4 a6

β2 a3 N6211

FUT8

β4 β2 a6 b4 b4

β4 β2 β2 β2 a6 b4 b4 LacZ a3 N211 a6 b4 b4 β4 β2 β2 a3 β2 a3 N001 a6 b4 b4 N000

β4 β2 a3 N111

Gn-T V

β6

β2 a6 b4 b4

β4 β2 a3 N3001

Figure 4.6.6 Scheme for the separation of N-glycan isomers from digestion of N001 with LacZ using FUT8 and Gn-T V.

A. N3001 -1 Galactose residue

β6 β2 a6 a6 b4 b4 b4 b4 β2 Gn-T V β4 β4 FUT8 X β4 a3 β4 a3 N37111 N3111 β2 β4 β2 β4 β4 β2 a6 Gn-T V b4 b4 FUT8 X β4 X a3 N3211

β4 β2

β4 β2 β4 β2 a6 Gn-T V a6 b4 b4 FUT8 b4 b4 a6 X β4 β4 β4 β4 a3 N3311 a3 N36311 β2 β2

B. N3001 -2 Galactose residue

β6 β4 β2 a6 b4 b4 a6 b4 β2 Gn-T V b4 β4 β4 FUT8 X a3 N37161 a3 N3161 β2 β2 β4 β4 β4 β2 β4 β2 a6 b4 b4 a6 a6 b4 b4 β4 Gn-T V β4 FUT8 X a3 N36261 a3 N3261 β2 β2

β6 β2 β2 a6 b4 b4 a6 b4 b4 a6 b4 b4 a6 β2 β4 β4 4 β4 Gn-T V β4 FUT8 β β4 a3 N37361 a3 N3361 a3 N36361 β2 2 β2 β

Figure 4.6.7 Scheme for LacZ digestion of N3001 and identification of possible isomer intermediate using FUT8 and Gn-T V. Reactions were monitored by MALDI-MS. A. N3001 -1 galactose residue possible isomers. B. N3001 -2 galactose residues possible isomers. Red box indicates the isomers identified from the MALDI-MS results (See pg.103 §4.6.11).

4.6.6 N-glycan labeling using 2-AA and AEAB

2-AA was used to labeled N001, N000, N111 and N211 for LacZ specificity studies.

AEAB was used to labeled N3001, N3000, N3111 and N3361 for Gn-T V specificity studies. 2-

AA or AEAB reactions were performed using the following conditions: 5 mg 2-AA or AEAB were dissolved in 100 µL solution of DMSO/AcOH (7:3 v/v). 6 mg NaCNBH3 were dissolved in the previous solution. 5 ul of labeling solution was added per every 200 µg glycan sample.

Labeling reaction was incubated in a water bath for 2 h at 65 °C. Labeled glycans were precipitated with acetronitrile (10 X the reaction volume) and incubated at -20 °C for 1 h.

Labeled glycans were centrifuged (13,000 g for 10 min), the supernatant was removed. The pellet was dried in a vacufuge for 15 min. Labeled glycan was diluted in ddH2O and analyzed by

MALDI-MS and HPLC-RF (2-AA :RF: excitation: 360 nm, emission: 420 nm, UV: 214 nm;

AEAB: RF: excitation: 330 nm, emission: 420 nm, UV: 330 nm).76 Sample concentration was determined using the peak area and comparing to 10 nmol known standard.

4.6.7 Substrate specificity studies on b-galactosidase (LacZ)

Substrate specificity assay of LacZ was performed against isomers N111 and N211 chemo- enzymatically synthesized N-glycans (with 2-AA label at reducing end) as acceptors (See Table

4.6.7.1). Reactions were performed in 10 µL total volume. Contains N-glycan substrate (50 µM),

Tris-HCl buffer (100 mM, pH 7.5), LacZ (1 mg/ml). Reactions were incubated at 37 °C overnight in a water bath. Samples were collected at 0, 5, 10, 15, 20, 30, 45 min, every hour for 8 h, and at

20 h. Reaction was quenched by freezing at 80 °C for 30 min. After centrifugation (13,000 g for

10 min), the supernatant was analyzed by HPLC using an analytical Waters XBridge BEH amide column (130 Å, 5 µm, 4.6 x 250 mm), detected by UV detector SPD-20A and a fluorescence detector RF-10Axl. UV absorption at 214 nm or fluorescence at 360nm excitation and 420nm emission. Gradient elution (%B: 65– 50%) was performed with Solvent A (100 mM ammonium formate, pH 3.2) and Solvent B (Acetonitrile). The percent yields (average of three replicated

99 reactions, see Table 4.6.7.1) were calculated as % = Product peak area/ (Product peak area +

Substrate peak area) × 100.

Table 4.6.7.1 Substrate specificity of LacZ. ND, Not Detected. Conversions were monitored by HPLC- RF, average of three replicates were shown. N111-AA to N000-AA N211-AA to N000-AA Time Conversion (%) Conversion (%) 0 min ND ND 5 min 4.1 ND 10 min 8.0 ND 15 min 9.9 ND 20 min 10.9 ND 30 min 16.7 ND 45 min 23.8 2.4 1 hr 30.9 3.8 2 hr 51.1 5.2 3 hr 65.3 8.2 4 hr 74.9 10.2 5 hr 83.1 11.9 6 hr 87.3 15.3 7 hr 90.5 16.7 8 hr 91.2 18.5 20 hr 100.0 28.6

4.6.8 Substrate specificity studies on Gn-T V

Gn-T V catalyzed reaction β6 β2 a6 b4 b4 a6 b4 b4 β2 β2 a3 N000 β2 a3 N7000

β6 β2 a6 b4 b4 a6 b4 b4 β2 β4 β4 a3 N3000 a3 N3700 β2 β2

Figure 4.6.8 Gn-T V catalyzed reaction for bi- and tri- antennary N-glycans.

A detailed substrate specificity study of Gn-T V was performed using 12 chemically or chemo-enzymatically synthesized N-glycans (with AEAB labeled reducing end) as acceptors

100

(Table 4.6.8.1) Reactions were performed in a 96 well PCR plate, each well (10 µL total volume) contains one N-glycan substrate (50 µM), UDP-GlcNAc (10 mM), MES buffer (100 mM, pH 7.0),

MgCl2 (15 mM), 0.2% Triton X-100, BSA (1 mg/ml), and Gn-T V (0.5 mg/mL). Reactions were incubated at 37 °C for 1 h in an Eppendorf thermocycler (Mastercycler Pro), followed by enzyme inactivation at 95 °C for 5 min. A negative control was also set up using N000 as substrate without enzyme. After centrifugation (13,000 g for 10 min), the supernatant was analyzed by HPLC using an analytical Waters XBridge BEH amide column (130 Å, 5 µm, 4.6 x 250 mm), detected by UV detector SPD-20A and a fluorescence detector RF-10Axl. UV absorption at 330 nm or fluorescence at 330nm excitation and 420nm emission. Gradient elution (%B: 65– 50%) was performed with Solvent A (100 mM ammonium formate, pH 3.2) and Solvent B (Acetonitrile).

The percent yields (average of three replicated reactions, see Table 4.6.8.1) were calculated as %

= Product peak area/ (Product peak area + Substrate peak area) × 100.

Table 4.6.8.1 Summary of specificity studies for Gn-T V. All of the substrates were AEAB labeled. * complete conversion after prolonged incubation. Glycan Substrate Conversion (%) N6000 61.2 ±7.5 N000 72.3 ±7.0 N020 ND N040 24.0 ±2.5 N050 45.6 ±4.1 N210 45.5 ±6.5 N110 ND N111 26.4 ±2.2 N112 10.6 ±2.1 N113 37.5 ±5.2 N114 29.7 ±5.7 N115 ND* N3001 ND N3000 19.6 ±1.0 N3111 13.8 ±1.4 N3361 18.4 ±3.4

101

4.6.9 Substrate specificity studies of Gn-T V on triantennary N-glycans

A substrate specificity study of Gn-T V was performed using 4 enzymatically synthesized tri-antennary N-glycans (with AEAB labeled reducing end) as acceptors (Table 4.6.9.1) Reactions were performed in PCR tubes (10 µL total volume) contains one N-glycan substrate (50 µM),

UDP-GlcNAc (10 mM), MES buffer (100 mM, pH 7.0), MgCl2 (15 mM), 0.2% Triton X-100,

BSA (1 mg/ml), and Gn-T V (0.5 mg/mL). Reactions were incubated at 37 °C for 1 h, overnight, and 3 days in an Eppendorf thermocycler (Mastercycler Pro) and water bath, followed by enzyme inactivation at 95 °C for 5 min. A negative control was also set up using N3000 as substrate without enzyme. After centrifugation (13,000 g for 10 min), the supernatant was analyzed by HPLC using an analytical Waters XBridge BEH amide column (130 Å, 5 µm, 4.6 x 250 mm), detected by UV detector SPD-20A and a fluorescence detector RF-10A xl. UV absorption at 330 nm or fluorescence at 330nm excitation and 420nm emission. Gradient elution (%B: 65– 50%) was performed with Solvent A (100 mM ammonium formate, pH 3.2) and Solvent B (Acetonitrile).

The percent yields (average of three replicated reactions, see Table 4.6.9.1) were calculated as %

= Product peak area/ (Product peak area + Substrate peak area) × 100.

Table 4.6.9.1 Gn-T V substrate specificity on triantennary N-glycans. ND, Not Detected. Conversions were monitored by HPLC-RF, average of three replicates were shown. Glycan Substrate Conversion 1 h (%) Conversion 3 days (%) N3001-AEAB ND ND N3000-AEAB 19.6 ±1.0 100 N3111-AEAB 13.8 ±1.4 100 N3361-AEAB 18.4 ±3.4 100

4.6.10 General methods for asymmetric glycan synthesis from natural products and

purification

Reactions catalyzed by LacZ, FUT8 and Gn-T V were performed and monitored by HPLC and

MALDI-MS. Digestion reactions by PNGaseF and Neuraminidase were performed as we

102 described previously. Digestion reactions by b-galactosidase (LacZ) were performed in 100 mM

Tris-HCl buffer (pH 7.5), containing 2 mM glycan acceptor and 1 mg/mL LacZ. Reactions were allowed to proceed for 3 h at 37 °C until substrates were totally converted to intermediate product

(monitored by HPLC-ELSD as described previously). The reaction condition for FUT8 was: N- glycan substrate (5 mM), GDP-fucose (10 mM), MES buffer (100 mM, pH 7.0), excess FastAP), and excess FUT8 (0.5 mg/mL). The reaction condition for Gn-T V was: N-glycan substrate (5 mM), UDP-GlcNAc (10 mM), MES buffer (100 mM, pH 7.0), MgCl2 (15 mM), 0.2% Triton X-

100, BSA (1 mg/ml), and excess Gn-T V (0.5 mg/mL). Reactions were incubated at 37 °C overnight or until substrates were totally converted to product. The reactions were then quenched by boiling for 5 min. After centrifugation (13,000 g for 10 min), the supernatant was analyzed.

HPLC-A210nm was then used to purify target glycans using a semi-preparative amide column (130

Å, 5 µm, 10 mm × 250 mm). The running conditions are solvent A: 100 mM ammonium formate

(for glycans with Neu5Ac residues) or water (for glycans without Neu5Ac residues), pH 3.2; solvent B: acetonitrile; flow rate: 4 mL/min; B%: 65-50% within 25 min. Products containing portions were then concentrated and lyophilized for characterization. The purity of each glycan was confirmed by HPLC-ELSD using an analytical Waters amide column (130 Å, 5 µm, 4.6 mm

× 250 mm). The running conditions are solvent A: 100 mM ammonium formate, pH 3.4; solvent

B: acetonitrile; flow rate: 1 mL/min; B%: 65-50% within 25 min. The molecular weight of each

N-glycans was confirmed by MALDI-MS performed on UltrafleXtreme MALDI TOF/TOF Mass

Spectrometer (Bruker). Scan range of MS1 was according to the molecular weight of N-glycans, and reflector mode was used for N-glycan analysis. Mass spectra were obtained in positive mode with the following voltage settings: ion source 1 (19.0 kV), ion source 2 (15.9 kV), and lens (9.3 kV). The reflector voltage was set to 20 kV. The laser was pulsed at 7 Hz and the pulsed ion

103 extraction time was set to 400 ns. The laser power was kept in the 25–40% range. Structures of key intermediates and asymmetric triantennary N-glycans were confirmed by 1H NMR.

4.6.11 HPLC profiles and MS data of purified N-glycans

N3001 (5 mg)

β4 β2 a6 b4 b4 β4 β4 a3

β4 β2

HPLC-ELSD, TR = 11.09 min

uV 2000

1750

1500

1250

1000

750

500

250

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 2005.7244; found [M+Na]+: 2028.7182

1200 2028.7182

1000 Intens. [a.u.] Intens. 800

600

400

200

0 1000 1200 1400 1600 1800 2000 m/z

104

N3111 (3.2 mg)

β2 a6 b4 b4 β4 β4 a3

β4 β2

HPLC-ELSD, TR = 10.06 min

uV 15000

12500

10000

7500

5000

2500

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 1843.6715; found [M+Na]+: 1866.6682

1000 1866.6682

800 Intens. [a.u.] Intens.

600

400

200

0 1000 1200 1400 1600 1800 2000 m/z

105

N37111 (0.2 mg)

β6

a6 b4 b4 β2 β4 β4 a3 β2 β4

HPLC-ELSD, TR = 10.86 min

400

350

300

250

200

150

100

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 2046.7509.; found [M+Na]+: 2069.7421

5000 2069.7421

Intens. [a.u.] Intens. 4000

3000

2000

1000

0 1000 1200 1400 1600 1800 2000 2200 2400 m/z

106

N3361 (0.2 mg)

β2 a6 b4 b4 β4 β4 a3 β2

HPLC-ELSD, TR = 8.87 min

7000

6000

5000

4000

3000

2000

1000

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 1681.6187.; found [M+Na]+: 1704.602

100 1704.602

80 Intens. [a.u.] Intens.

0 1000 1200 1400 1600 1800 2000 2200 m/z

107

N37361 (0.1 mg)

β6

a6 b4 b4 β2 β4 β4 a3 β2

HPLC-ELSD, TR = 14.91min

300

275

250

225

200

175

150

125

100

-25

7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1884.6981.; found [M+Na]+: 1907.6851

1907.6851 125

Intens. [a.u.] Intens. 100

0 1000 1200 1400 1600 1800 2000 2200 2400 m/z

108

N36361 (0.1 mg)

β2 a6 b4 b4 a6 β4 β4 a3 β2

HPLC-ELSD, TR = 13.97 min, 65-50 % gradient

350

300

250

200

150

100

7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1827.6766.; found [M+Na]+: 1850.6662

1850.6662 800

Intens. [a.u.] Intens. 600

400

200

0 1000 1200 1400 1600 1800 2000 m/z

109

N3000 (0.2 mg)

β2 a6 b4 b4 β4 a3 β2

HPLC-ELSD, TR = 7.65 min

2500

2000

1500

1000

500

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 1519.5659.; found [M+Na]+: 1542.5548

1542.5548 6000

Intens. [a.u.] Intens. 5000

4000

3000

2000

1000

0 1400 1500 1600 1700 1800 1900 m/z

110

N37000 (0.1 mg)

β6

a6 b4 b4 β2 β4 a3 N37000 β2

MALDI-MS, calculated: 1722.6453.; found [M+Na]+: 1745.6322

1500 1745.6322

1250 Intens. [a.u.] Intens.

1000

750

500

250

0 1000 1200 1400 1600 1800 2000 2200 2400 m/z

111

N36000 (0.1 mg)

β2 a6 b4 b4 a6 β4 a3 N36000 β2

MALDI-MS, calculated: 1665.6238.; found [M+Na]+: 1688.6191

1688.6191

Intens. [a.u.] Intens. 30

0 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 m/z

112

4.6.12 NMR spectra and data of purified asymmetric biantennary and triantennary N-glycans

N3001

β4 β2 a6 b4 b4 β4 β4 a3

β4 β2

To establish the linkage profile for the N3001, 1H NMR, 13C NMR, and multiple 2D-

NMR experiments were performed to enable careful structural analysis achieving signal assignments and observation of key correlations. Briefly, the combination of HMBC and HSQC-

TOCSY spectra displayed 5 critical three-bond C-H correlations, which revealed the structure as the one having 2 antennae at C-3 branch and 1 antenna at C-6 branch.

1H NMR

1 H NMR (600 MHz, D2O) δ 5.09 (s, 0.6H, H-1 GlcNAc-1α), 5.01 (s, 1H, H-1 Man-4), 4.82 (s,

1H, H-1 Man-4’), 4.66 (s, 1H, H-1 Man-3), 4.60 (d, J = 7.6 Hz, 0.4H, H-1 GlcNAc-1β), 4.53 –

4.43 (m, 4H, H-1 GlcNAc-2, 5, 7, and 7’), 4.40 – 4.32 (m, 3H, H-1 Gal-6, 8, and 8’), 4.16 – 4.07

(m, 2H, H-2 Man-4 and 3), 4.01 (s, 1H, H-2 Man-4’), 3.95 (d, J = 9.3 Hz, 1H), 3.92 – 3.36 (m,

62H), 2.04 – 1.87 (m, 15H, NHAc).

113

HSQC spectrum

HMBC spectrum

114

HSQC-TOCSY spectrum

N3111

β2 a6 b4 b4 β4 β4 a3

β4 β2

1H NMR and multiple 2D-NMR experiments were performed for the structural elucidation to confirm the glycosidic linkage profile. Essentially, the combination of HMBC and

HSQC-TOCSY spectra indicated the structure as the one shown in the scheme (the cleaved Gal situated on upper branch).

115

1H NMR

1 H NMR (600 MHz, H2O+D2O) δ 5.13 (s, 0.5H, H-1 GlcNAc-1α), 5.05 (s, 1H, H-1 Man-4), 4.85

(s, 1H, H-1 Man-4’), 4.70 (s, 1H, H-1 Man-3), 4.63 (d, J = 6.6 Hz, 0.5H, H-1 GlcNAc-1β), 4.58

– 4.45 (m, 4H, H-1 GlcNAc-2, 5, 7, and 7’), 4.44 – 4.35 (m, 2H, H-1 Gal-6 and 8), 4.15 (m, 2H,

H-2 Man-4 and 3), 4.05 (s, 1H, H-3 Man-4), 3.99 (d, J = 8.3 Hz, 1H, H-2 Man-4’), 3.96 – 3.89

(m, 3H, H-4 Gal-6 and 8, H-2 GlcNAc-1α), 3.89 – 3.80 (m, 8H), 3.80 – 3.29 (m, 45H), 2.08 –

1.91 (m, 15H, NHAc).

116

HSQC spectrum

HMBC spectrum

117

HSQC-TOCSY spectrum

N211

β4 β2 a6 b4 b4

β2 a3

1H NMR and HSQC experiments were performed in order to confirm the glycosidic linkage profile. However, structural elucidation could not been achieved without HMBC and

HSQC-TOCSY spectra. Alternatively, the structure was revealed as the one bearing a Gal residue at the C-6 branch by comparison with previously reported spectrum.

118

1H NMR

1H NMR (600 MHz, H2O+D2O) δ 5.12 (s, 0.6H, H-1 GlcNAc-1α), 5.04 (s, 1H, H-1 Man-4), 4.86

(s, 1H, H-1 Man-4’), 4.70 (s, 1H, H-1 Man-3), 4.63 (d, J = 7.6 Hz, 0.4H, H-1 GlcNAc-1β), 4.56 –

4.46 (m, 3H, H-1 GlcNAc-2, 5, and 5’), 4.41 (d, J = 7.5 Hz, 1H, H-1 Gal), 4.18 (s, 1H), 4.12 (s,

1H), 4.04 (s, 1H), 3.95 – 3.33 (m, 45H), 2.11 – 1.91 (m, 12H, NHAc).

HSQC spectrum

119

4.7 References

1. A. Varki and J. B. Lowe, in Essentials of Glycobiology, eds. A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart and M. E. Etzler, Cold Spring Harbor (NY), 2nd edn., 2009. 2. Y. Y. Zhao, M. Takahashi, J. G. Gu, E. Miyoshi, A. Matsumoto, S. Kitazume and N. Taniguchi, Cancer science, 2008, 99, 1304-1310. 3. R. A. Dwek, Chemical reviews, 1996, 96, 683-720. 4. B. Imperiali and S. E. O'Connor, Current opinion in chemical biology, 1999, 3, 643-649. 5. A. Helenius and M. Aebi, Science, 2001, 291, 2364-2369. 6. A. Helenius and M. Aebi, Annual review of biochemistry, 2004, 73, 1019-1049. 7. I. Prahl and C. Unverzagt, Tetrahedron Letters, 2000, 41, 10189-10193. 8. S. Eller, R. Schuberth, G. Gundel, J. Seifert and C. Unverzagt, Angewandte Chemie, 2007, 46, 4173-4175. 9. D. Ott, J. Seifert, I. Prahl, M. Niemietz, J. Hoffman, J. Guder, M. Mönnich and C. Unverzagt, European Journal of Organic Chemistry, 2012, 2012, 5054-5068. 10. B. Sun, B. Srinivasan and X. Huang, Chemistry, 2008, 14, 7072-7081. 11. S. Serna, S. Yan, M. Martin-Lomas, I. B. Wilson and N. C. Reichardt, Journal of the American Chemical Society, 2011, 133, 16495-16502. 12. S. Q. Fan, W. Huang and L. X. Wang, J Biol Chem, 2012, 287, 11272-11281. 13. W. Huang, J. Giddens, S. Q. Fan, C. Toonstra and L. X. Wang, Journal of the American Chemical Society, 2012, 134, 12308-12318. 14. L. Li, Y. Liu, C. Ma, J. Qu, A. D. Calderon, B. Wu, N. Wei, X. Wang, Y. Guo, Z. Xiao, J. Song, G. Sugiarto, Y. Li, H. Yu, X. Chen and P. G. Wang, Chemical science, 2015, 6, 5652-5661. 15. K. R. Anumula, Anal Biochem, 2000, 283, 17-26. 16. K. R. Anumula and S. T. Dhume, Glycobiology, 1998, 8, 685-694. 17. D. J. Harvey, PROTEOMICS, 2001, 1, 311-328. 18. S. Hua, H. J. An, S. Ozcan, G. S. Ro, S. Soares, R. DeVere-White and C. B. Lebrilla, The Analyst, 2011, 136, 3663-3671. 19. Y. Huang, Y. Nie, B. Boyes and R. Orlando, Journal of Biomolecular Techniques : JBT, 2016, 27, 98-104. 20. Y. Mechref, Electrophoresis, 2011, 32, 3467-3481. 21. Y. Pu, M. E. Ridgeway, R. S. Glaskin, M. A. Park, C. E. Costello and C. Lin, Analytical Chemistry, 2016, 88, 3440-3443. 22. I. Rustighi, C. Campa, M. Rossi, S. Semeraro, A. Vetere and A. Gamini, Electrophoresis, 2009, 30, 2632-2639. 23. Z. Szabo, A. Guttman, T. Rejtar and B. L. Karger, Electrophoresis, 2010, 31, 1389-1395. 24. J. P. Williams, M. Grabenauer, R. J. Holland, C. J. Carpenter, M. R. Wormald, K. Giles, D. J. Harvey, R. H. Bateman, J. H. Scrivens and M. T. Bowers, International Journal of Mass Spectrometry, 2010, 298, 119-127. 25. A. D. Calderon, Y. Liu, X. Li, X. Wang, X. Chen, L. Li and P. G. Wang, Org Biomol Chem, 2016, 14, 4027-4031. 26. R. D. Cummings, I. S. Trowbridge and S. Kornfeld, J Biol Chem, 1982, 257, 13421- 13427.

120

27. K. L. Abbott, K. Aoki, J. M. Lim, M. Porterfield, R. Johnson, R. M. O'Regan, L. Wells, M. Tiemeyer and M. Pierce, J Proteome Res, 2008, 7, 1470-1480. 28. K. L. Abbott, A. V. Nairn, E. M. Hall, M. B. Horton, J. F. McDonald, K. W. Moremen, D. M. Dinulescu and M. Pierce, Proteomics, 2008, 8, 3210-3220. 29. B. Fernandes, U. Sagman, M. Auger, M. Demetrio and J. W. Dennis, Cancer Res, 1991, 51, 718-723. 30. T. Handerson, R. Camp, M. Harigopal, D. Rimm and J. Pawelek, Clin Cancer Res, 2005, 11, 2969-2973. 31. K. Murata, E. Miyoshi, M. Kameyama, O. Ishikawa, T. Kabuto, Y. Sasaki, M. Hiratsuka, H. Ohigashi, S. Ishiguro, S. Ito, H. Honda, F. Takemura, N. Taniguchi and S. Imaoka, Clin Cancer Res, 2000, 6, 1772-1777. 32. W. K. Seelentag, W. P. Li, S. F. Schmitz, U. Metzger, P. Aeberhard, P. U. Heitz and J. Roth, Cancer Res, 1998, 58, 5559-5564. 33. H. Tian, E. Miyoshi, N. Kawaguchi, M. Shaker, Y. Ito, N. Taniguchi, M. Tsujimoto and N. Matsuura, Pathobiology, 2008, 75, 288-294. 34. M. Nonaka, J Gastroenterol, 2016, 51, 406-408. 35. B. Li, S. Su, M. Y. Zhang, L. He, Q. D. Wang and K. He, Mol Med Rep, 2016, 13, 469- 476. 36. J. B. Wu, L. Shen, L. Qiu, Q. W. Duan, Z. G. Luo and X. X. Dong, Int J Clin Exp Pathol, 2015, 8, 9901-9911. 37. B. Huang, L. Sun, J. Cao, Y. Zhang, Q. Wu, J. Zhang, Y. Ge, L. Fu and Z. Wang, Oncol Rep, 2013, 29, 2392-2400. 38. E. Miyoshi, M. Terao and Y. Kamada, BMB Rep, 2012, 45, 554-559. 39. A. Kyan, N. Kamimura, S. Hagisawa, S. Hatakeyama, T. Koie, T. Yoneyama, Y. Arai, H. Nakagawa, S. Nishimura, E. Miyoshi, Y. Hashimoto and C. Ohyama, Int J Oncol, 2008, 32, 129-134. 40. S. Nakahara, T. Saito, N. Kondo, K. Moriwaki, K. Noda, S. Ihara, M. Takahashi, Y. Ide, J. Gu, H. Inohara, T. Katayama, M. Tohyama, T. Kubo, N. Taniguchi and E. Miyoshi, FASEB J, 2006, 20, 2451-2459. 41. L. Y. Wang, K. L. Chen, J. M. Su, J. W. Jin, H. L. Chen and X. L. Zha, Shi Yan Sheng Wu Xue Bao, 2001, 34, 219-225. 42. N. Taniguchi, S. Ihara, T. Saito, E. Miyoshi, Y. Ikeda and K. Honke, Glycoconj J, 2001, 18, 859-865. 43. M. Demetriou, M. Granovsky, S. Quaggin and J. W. Dennis, Nature, 2001, 409, 733-739. 44. S. Ihara, E. Miyoshi, J. H. Ko, K. Murata, S. Nakahara, K. Honke, R. B. Dickson, C. Y. Lin and N. Taniguchi, J Biol Chem, 2002, 277, 16960-16967. 45. S. Ihara, E. Miyoshi, S. Nakahara, H. Sakiyama, H. Ihara, A. Akinaga, K. Honke, R. B. Dickson, C. Y. Lin and N. Taniguchi, Glycobiology, 2004, 14, 139-146. 46. E. A. Partridge, C. Le Roy, G. M. Di Guglielmo, J. Pawling, P. Cheung, M. Granovsky, I. R. Nabi, J. L. Wrana and J. W. Dennis, Science, 2004, 306, 120-124. 47. T. Saito, E. Miyoshi, K. Sasai, N. Nakano, H. Eguchi, K. Honke and N. Taniguchi, J Biol Chem, 2002, 277, 17002-17008. 48. P. Cheung and J. W. Dennis, Glycobiology, 2007, 17, 767-773. 49. J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman and R. S. Kerbel, Science, 1987, 236, 582-585.

121

50. M. Granovsky, J. Fata, J. Pawling, W. J. Muller, R. Khokha and J. W. Dennis, Nat Med, 2000, 6, 306-312. 51. H. B. Guo, H. Johnson, M. Randolph, T. Nagy, R. Blalock and M. Pierce, Proc Natl Acad Sci U S A, 2010, 107, 21116-21121. 52. P. Buckhaults, L. Chen, N. Fregien and M. Pierce, J Biol Chem, 1997, 272, 19575-19581. 53. L. Chen, W. Zhang, N. Fregien and M. Pierce, Oncogene, 1998, 17, 2087-2093. 54. R. Kang, H. Saito, Y. Ihara, E. Miyoshi, N. Koyama, Y. Sheng and N. Taniguchi, J Biol Chem, 1996, 271, 26706-26712. 55. J. H. Ko, E. Miyoshi, K. Noda, A. Ekuni, R. Kang, Y. Ikeda and N. Taniguchi, J Biol Chem, 1999, 274, 22941-22948. 56. I. Brockhausen, J. P. Carver and H. Schachter, Biochem Cell Biol, 1988, 66, 1134-1151. 57. I. Brockhausen, S. Narasimhan and H. Schachter, Biochimie, 1988, 70, 1521-1533. 58. T. Isaji, J. Gu, R. Nishiuchi, Y. Zhao, M. Takahashi, E. Miyoshi, K. Honke, K. Sekiguchi and N. Taniguchi, J Biol Chem, 2004, 279, 19747-19754. 59. K. Sasai, Y. Ikeda, H. Eguchi, T. Tsuda, K. Honke and N. Taniguchi, FEBS Lett, 2002, 522, 151-155. 60. Y. Sato, T. Isaji, M. Tajiri, S. Yoshida-Yamamoto, T. Yoshinaka, T. Somehara, T. Fukuda, Y. Wada and J. Gu, J Biol Chem, 2009, 284, 11873-11881. 61. M. Yoshimura, A. Nishikawa, Y. Ihara, S. Taniguchi and N. Taniguchi, Proc Natl Acad Sci U S A, 1995, 92, 8754-8758. 62. A. Nishikawa, J. Gu, S. Fujii and N. Taniguchi, Biochim Biophys Acta, 1990, 1035, 313- 318. 63. M. M. Palcic, J. Ripka, K. J. Kaur, M. Shoreibah, O. Hindsgaul and M. Pierce, J Biol Chem, 1990, 265, 6759-6769. 64. M. Pierce, J. Arango, S. H. Tahir and O. Hindsgaul, Biochem Biophys Res Commun, 1987, 146, 679-684. 65. D. J. Wasilko, S. E. Lee, K. J. Stutzman-Engwall, B. A. Reitz, T. L. Emmons, K. J. Mathis, M. J. Bienkowski, A. G. Tomasselli and H. D. Fischer, Protein expression and purification, 2009, 65, 122-132. 66. Y. Zou, Z. Wu, L. Chen, X. Liu, G. Gu, M. Xue, P. G. Wang and M. Chen, Journal of Carbohydrate Chemistry, 2012, 31, 436-446. 67. B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver and D. A. Cumming, Biochemistry, 1989, 28, 6491-6499. 68. D. Witzigmann, P. Detampel, F. Porta and J. Huwyler, RSC Advances, 2016, 6, 97636- 97640. 69. E. Berman and P. Bendel, FEBS Lett, 1986, 204, 257-260. 70. S. Takasaki and A. Kobata, Biochemistry, 1986, 25, 5709-5715. 71. R. R. Townsend, M. R. Hardy, T. C. Wong and Y. C. Lee, Biochemistry, 1986, 25, 5716- 5725. 72. O. Renkonen, J. Helin, A. Vainio, R. Niemela, L. Penttila and P. Hilden, Biochem Cell Biol, 1990, 68, 1032-1036. 73. D. H. van den Eijnden, W. M. Blanken and A. van Vliet, Carbohydr Res, 1986, 151, 329- 335. 74. G. Zhao, W. Guan, L. Cai and P. G. Wang, Nature protocols, 2010, 5, 636-646. 75. H. Ihara, Y. Ikeda and N. Taniguchi, Glycobiology, 2006, 16, 333-342.

122

76. X. Song, B. Xia, S. R. Stowell, Y. Lasanajak, D. F. Smith and R. D. Cummings, Chemistry & biology, 2009, 16, 36-47.

123

5 CONCLUSIONS

TIPs method successfully allowed us to express, purify and characterize five hGTs (Gn-T

I, Gn-T II, Gn-T III Gn-T V, and FUT8) in large scale from baculovirus-infected Sf9 cells. Each of the enzymes was purified and their activities assayed. All of the enzymes were active with the exception of Gn-T III which show very low activity.

Human FUT8 substrate specificity was studied in detailed and the results concluded that there is a strict requirement towards the a1,3-mannose branch, and very relax requirements towards the a1,6-mannose branch. human FUT8 can also core-fucosylated an N-glycan in vitro without the a1,3-mannose branch, which was not observed before. In addition, a facile chemo- enzymatic strategy was developed and employed for the efficient synthesis of a panel of a1,6 core- fucosylated asymmetric N-glycans.

Human Gn-T V substrate specificity was studied in detailed and the results concluded that there is a strict requirement towards the a1,6-mannose branch, and very relax requirements towards the a1,3-mannose branch. A novel strategy for the enzymatic separation of bi- and tri- antennary asymmetric N-glycan isomers was successfully developed, which takes advantage of the substrate specificity requirements of well-studied human glycosyltransferase FUT8 and Gn-T

V. Large scale synthesis and separation of asymmetric N-glycans from natural sources is highly desirable for the generation of well-defined asymmetric N-glycan structures valuable as standards and probes for glycomic analysis and elucidation of glycan-binding protein functions.

These new chemo-enzymatic strategies will play a crucial role in the synthesis of potential antigenic N-glycans for future glycoconjugate vaccines.

124

APPENDICES

Appendix A Additional human glycosyltransferases expressed in Sf9 using TIPs method.

(Gn-T I, Gn-T II, and Gn-T III)

Appendix A.1 SDS-PAGE for Gn-T I and Gn-T II

Gn-T I Gn-T II kDa - 170 - 100 - 70 - 55 - 40 - 35 - 25

- 10

Appendix A.2 Western-Blot for Gn-T III

kDa Gn-T III 170 - 100 - 70 - 55 - 40 - 35 - 25 -

10 -

125

Appendix A.3 Activity Assay for Gn-T I, Gn-T II and Gn-T III

Gn-T I Activity assay

a6 a6

a3 a3 b2

ESI-MS, calculated acceptor: 910.3278; found [M+H]+: 912.3664; calculated product:

1113.4072; found [M+H]+: 1114.4460 [M+Na]+: 1137.4274.

NL: 1114.4460 z=1 100 95 90 85 80 912.3664 75 z=1 70

65 60 55 50 45 40 Relative Abundance 1121.4545 35 952.0327 z=2 z=1 30 899.1107 z=1 25 20 946.2469 z=1 982.0009 1137.4274 z=1 z=1 15 890.1512 921.0921 1020.1844 z=1 z=1 991.1586 z=1 1190.1314 10 z=1 870.9847 1039.9745 1073.1067 1159.1914 z=1 z=1 965.2892 1009.9914 z=1 5 z=1 z=1 1097.4369 z=1 z=1 z=2 0 880 900 920 940 960 980 1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 m/z

Reaction conditions: Substrate (1.0 mM), UDP-GlcNAc (4.0 mM), HEPES pH 7.0 (20 mM),

MnCl2 (20 mM), Gn-T II (0.1 mg/ml)

126

Gn-T II Activity assay

a6 b2 a6

b2 b2 a3 a3

ESI-MS, calculated acceptor: 1113.4072; found [M+H]+: 1114.4151; calculated product:

1316.4865; found [M+H]+: 1317.4938, [M+Na]+: 1339.4754.

Angei_G2P5 #164-252 RT: 1.45-2.13 AV: 44 NL: 5.66E3 T: FTMS + p ESI Full ms [400.00-2000.00] 1317.4938 z=1 100

75 1114.4151 70 z=1

50 1339.4754 z=1 45

RelativeAbundance 40

35 1167.3377 z=1 30 1344.4567 25 z=2 1370.4164 20 1136.3969 z=1 z=1 15 1096.4052 10 z=1 1314.5305 1160.1266 1221.9914 z=1 z=? 1421.9780 5 z=? 1276.4673 1400.9118 1440.7081 1489.3493 1175.4703 z=5 z=2 z=? z=1 z=? z=? 0 1100 1150 1200 1250 1300 1350 1400 1450 1500 m/z

Reaction conditions: Substrate (1.0 mM), UDP-GlcNAc (4.0 mM), HEPES pH 7.0 (20 mM),

MnCl2 (20 mM), Gn-T II (0.1 mg/ml).

127

Gn-T III Activity assay

b2 b2 a6 a6 b4 b2 a3 a3 b2

ESI-MS, calculated acceptor: 1316.4865; found [M+Na]+: 1340.419; calculated product:

1519.5659; found [M+Na]+: 1543.219

033016 0:I1 MS Raw Intens. [a.u.] Intens.

5000

1486.574

4000

3000 1340.419

2000

1000

1543.219

0 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z

Reaction conditions: Substrate (1.0 mM), UDP-GlcNAc (5.0 mM), MES pH 6.5 (100 mM),

MnCl2 (10 mM), Gn-T II (0.1 mg/ml)

Appendix B Additional chemo-enzymatic synthesized a1,6-core-fucosylated N-glycan structures

The following core-fucosylated structures were synthesized for a collaboration project with Dr. Linda Hsieh-Wilson from CalTech. The project aims to develop a chemo-enzymatic approach to label core-fucosylated glycans using galactosyltransferase (GALT-1), which

128 transfers a labeled galactose residue to the a1,6-core fucose of N-glycans. The synthesized CF- glycans provided them with well-defined structures for GALT-1 substrate specificity studies.

Appendix B.1 Scheme for chemo-enzymatic synthesis of additional complex symmetric

core-fucosylated N-glycans.

Scheme for complex symmetric CF-glycans chemo-enzymatic synthesis

β2 β2 β4 β2 a3 β4 β2 a6 a6 a6 b4 b4 FUT8 a6 b4 b4 a6 bGalT b4 b4 a6 PmST1 b4 b4 a6

β2 a3 β2 a3 β4 β2 a3 a3 β4 β2 a3 N6000 N6001 N000 N6002

Appendix B.2 HPLC profiles and MS data of purified complex symmetric N-glycans

N000 (3 mg)

β2 a6 b4 b4

β2 a3

MALDI-MS, calculated: 1316.4865; found [M+Na]+: 1339.496

N6000 (1 mg)

129

β2 a6 b4 b4 a6

β2 a3

HPLC-ELSD, TR = 11.57 min

5000

4000

3000

2000

1000

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

MALDI-MS, calculated: 1462.5444; found [M+Na]+: 1485.5155

1485.5155 8000 Intens. [a.u.] Intens. 6000

4000

2000

0 800 1000 1200 1400 1600 1800 m/z

130

N6001 (0.5 mg)

β4 β2 a6 b4 b4 a6

β4 β2 a3

HPLC-ELSD, TR = 15.30 min

1250

1000

750

500

250

7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

MALDI-MS, calculated: 1786.6501; found [M+Na]+: 1809.6393

4 x10 1809.6033

1.0 Intens. [a.u.] Intens.

0.8

0.6

0.4

0.2

0.0 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z

131

N6002 (0.5 mg)

a3 β4 β2 a6 b4 b4 a6

a3 β4 β2 a3

HPLC-ELSD, TR = 17.17 min

uV 500

400

300

200

100

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mi n

ESI-MS, calculated: 2368.8409; found [M-H]-: 2367.7881, [M-2H]-2: 1183.8853

Angie_N6002(1)_033016 #122-298 RT: 1.58-3.84 AV: 166 NL: 5.85E3 T: FTMS - p ESI Full ms [500.00-3000.00] 2367.7881 z=1 100

65 1183.8853 z=2 60

RelativeAbundance 40

20 2389.7692 15 1160.5038 z=1 z=? 1212.3615 10 z=2 2400.7821 1357.8899 z=1 1693.7235 5 1011.4324 z=? 1549.8612 1770.4741 2021.2885 2135.1742 2252.6257 2502.3332 2695.3168 2886.9182 z=2 z=? z=2 z=? z=? z=? z=? z=? z=? z=1 0 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z