Structural Studies of Oligosaccharides Attached to Proteins Expressed in Different Organisms and Pegylation of a Non-Glycosylated Protein

Structural Studies of Oligosaccharides Attached to Proteins Expressed in Different Organisms and PEGylation of a non-Glycosylated Protein

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Edwin Mwamba Motari, M.S.

Graduate Program in Chemistry

The Ohio State University

2010

Dissertation Committee:

Professor Peng G. Wang, Advisor

Professor Michael A. Freitas

Professor Heather Allen

Edwin Mwamba Motari

2010

Abstract

Glycosylation is an important modification in proteins in that it will signal for proper folding of proteins. It also has several important roles in the cell for example structural roles like aiding in stability of proteins; they act as recognition epitopes of the proteins that they are attached to. Oligosaccharides on protein on cell surface also act as receptors of viruses, bacteria and toxins thus acting as the entry point of harmful agents into the body. Therefore the study of the structure of these compounds is important in understanding their roles and functions. To mimic this type of modification PEGylation has been used in therapeutic proteins. PEGylation helps mainly in increasing the half-life of these compounds in the body and also increase the solubility and stability of protein drugs.

In this research analytical methods were used to determine the glycans structure on recombinant glycoprotein CD24. In chapter 2, the average structures of glycans on

CD24 were determined by the use of MALDI-TOF-MS after the glycans were released with chemicals means and enzymatic means. The result showed the presence of both N- and O-glycans with the major compound being the O-glycans. The O-glycans that mostly expressed was found to be the sialyl T-antigen. To determine the glycosylation sites on the protein one N-glycosylation site on the fusion portion was determined on the amino peptide, EEQYNSTYR after trypsin digestion.

iii

In chapter four, the glycosylation study of N-glycans from a recombinant protein

TNFR-Fc expressed in the plant Camelina sativa was carried. The N-glycans were released with PNGase A and PNGase F. The results revealed the presence of plant-like glycans due to the presence of Xylose and α-1,3 Fucose and animal-like N-glycans.

In the last chapter a variant of paraoxonase 1 -a catalytic bioscavenger against organophosphate compounds was expressed in E. coli and PEGylated with a 28 kDa NHS activated mPEG. PEGylation is an important protein modification in improving its solubility and serum half-life. After the PEGylation, the PEGylated protein was purified by anion exchange chromatography and the kinetic studies for this enzyme against paraoxon carried out. The result revealed a reduction of the Kcat/Km values when compared to the unPEGylated enzyme.

Dedication

This document is dedicated to my parents.

Acknowledgments

I would like to give my sincere thanks to my adviser Dr. Peng G. Wang for his guidance during the research. His continued provision of advice during the research was very helpful towards the attaining of the results obtained. I also thank all my group members for the help that they provided especially Kaarina Lokko in the PEGylation of paraoxonase 1, Dr. Yalong Zhang in the use of MS in our lab and PEgylation and Dr. Yi

Wen in analysis of protein.

I express my appreciation to Dr. Yang Liu for providing the recombinant CD24 that was used in this study and with Dr. Xincheng Zhang in their helping me to understand the importance of the glycoprotein.

I also sincerely thank Dr. Mike Freitas for the guidance in the use of mass spectrometry for the analysis of protein modification and data analysis. I also thank his group members Dr. Xiaodan Su, Dr. Lanhao Yang, Kelly DiRienzo and the other lab members for helping me in running the LC-MS-MS in their laboratory.

I also acknowledge Dr Mamuka Kvaratskelia and his lab in helping me run my samples in the MALDI-TOF-MS and the help that they provided in analyzing the data.

I also thank Dr. Malgiery Lab members Dr. Vivek Shete and Christina Harsch for their help in paraoxonase 1 expression and I also thank Dr. Richard Sayre and his lab member Dr. Vanessa Falcao for providing the Fea1 that was used in this study. I thank

vi the Campus Chemical Instrument Center (CCIC) at The Ohio State University especially

Dr. Kari Green-Church, Dr. Liwen Zhang and the other staff members in helping with

LC-MS-MS and GC-MS analysis of my samples.

Finally I thank my family and all my friends who have always been there for me during the entire period of this study.

vii

Vita

March 1999 ...... B.S.Chemistry, Jomo Kenyatta University of

Agriculture and Technology

1999-2002 ...... Quality analyst Regal Pharmaceuticals

2002-2004 ...... M.S. Chemistry, Indiana University of

Pennsylvania

2004-2006 ...... Graduate Teaching Associate, Department

of Chemistry, The Ohio State University

2006 to present ...... Graduate Research Associate, Department

of Chemistry, The Ohio State University

Publications

1 Yi Wen; Yao Qingjia; Zhang Yalong; Motari Edwin; Lin Steven; Wang Peng

George The wbnH gene of Escherichia coli O86:H2 encodes an alpha-1,3-N-

acetylgalactosaminyl transferase involved in the O-repeating unit

biosynthesis.Biochemical and biophysical research communications (2006),

344(2), 631-9

2 Yi, Wen; Perali, Ramu; Eguchi, Hironobu; Motari, Edwin; Woodward, Robert;

Wang, P. George Characterization of a Bacterial β-1,3-Galactosyltransferase

viii

With Application in the Synthesis of Tumor-Associated T-Antigen Mimics"

Biochemistry Biochemistry, 2008, 47 (5), pp 1241-1248.

3 Jon O. Nagy, Yalong Zhang, Wen Yi, Xianwei Liu, Edwin Motari, Jing C. Song,

Jeffery T. Lejeune and Peng G. Wang, Glycopolydiacetylene nanoparticles as

a chromatic biosensor to detect Shiga-like toxin producing Escherichia coli

O157:H7, Bioorg Med Chem Lett. 2008 Jan 15;18(2):700-3.

4 Edwin Motari, Xincheng Zheng, Xiaodan Su, Yang Liu, Mamuka Kvaratskhelia,

Michael Freitas, Peng G. Wang, Analysis of Recombinant CD24 Glycans by

MALDI-TOF-MS Reveals Prevalence of Sialyl-T Antigen, Am. J. Biomed.

Sci. 2009, 1, 1- 11.

5 Woodward, R., Yi, W., Li, L., Zhao, G., Eguchi, H., Sridhar, P.R., Guo, H., Song,

J.K., Motari, E., Cai, L., Kelleher, P., Liu, X., Han, W., Zhang, W., Ding, Y.

and Wang, P.G. In vitro bacterial polysaccharide biosynthesis: Defining the

functions for Wzy and Wzz. Nat. Chem. Biol. accepted.

Fields of Study

Major Field: Chemistry

Analytical Chemistry

Table of Contents

Abstract ...... iii

Dedication ...... v

Acknowledgments...... vi

Vita ...... viii

List of Tables ...... xx

List of Figures ...... xxi

CHAPTER 1 : Glycan s structure analysis of glycoprotein and protein PEGylation ...... 1

1.1Glycosylation introduction ...... 1

1.1.1 Protein glycosylation ...... 2

1.1.2 Mammalian N-linked glycoprotein biosynthesis ...... 3

1.1.2.1 Types of N-linked oligosaccharides ...... 4

1.1.3 Biosynthesis of O-linked glycoproteins ...... 5

1.1.4 Biological functions of glycoproteins...... 7

1.1.5 Protein glycosylation and disease ...... 9

1.1.6 Therapeutic glycoproteins and carbohydrates ...... 11

1.2 Analytical Methods for Glycoprotein and Glycan Characterization ...... 13

1.2.1 Intact glycoproteins analysis ...... 13

1.2.2 Glycopeptide analysis and site-specific glycosylation analysis ...... 14

1.2.3 Cleavage of glycans form glycoproteins: chemical and enzymatic ...... 15

1.2.3.1 Chemical cleavage ...... 15

1.2.3.1.1. Hydrazinolysis: ...... 15

1.2.3.1.2. Alkaline β-elimination ...... 16

1.2.3.2 Enzymatic cleavage ...... 17

1.2.4 Analysis of the released glycan mixtures...... 18

1.2.4.1 Mass Spectrometry...... 18

1.2.4.1.1 FAB-MS ...... 18

1.2.4.1.2 ESI-MS ...... 19

1.2.4.1.3 MALDI-MS ...... 20

1.2.4.2 Chromatography ...... 21

1.2.4.2.1 Use of reverse-phase (RP) columns ...... 21

1.2.4.2.2 Use of normal-phase (NP) columns ...... 22

1.2.4.2.3 Use of Graphitized carbon columns ...... 23

1.2.4.2.4 High-Performance Anion-Exchange Chromatography (HPAEC) ...... 24

1.2.4.2.5 Use of Lectin Affinity columns ...... 24

1.2.4.2.6 Capillary Electrophoresis ...... 26

1.2.4.2.7 Fluorophore-Assisted carbohydrate electrophoresis (FACE) ...... 27

1.3 Protein modification to mimic glycosylation by PEGylation ...... 28

1.3.1 PEGylation introduction ...... 28

1.3.2 Importance of PEGylation of therapeutic proteins ...... 29

1.3.3 Chemistry of PEGylation...... 29

1.3.3.1 PEG chemistry for conjugation to amine ...... 29

1.3.3.2 PEG chemistry for cysteine modification ...... 31

1.3.3.3 PEG chemistry for oxidized carbohydrates or N-terminus ...... 32

1.3.3.4 Heterobifunctional PEG chemistry ...... 33

1.3.3.5 PEGylation using dock and lock (DNL) technology ...... 33

1.4 Research goals ...... 35

CHAPTER 2 The glycosylation analysis of recombinant CD24 ...... 49

2.1 CD24 protein introduction ...... 49

2.2 Experimental ...... 51

2.2.1 Materials ...... 51

2.2.2 Chemical cleavage by beta-elimination ...... 51

2.2.3 Cleavage of CD24 from CD24IgG1 with papain ...... 51

2.2.4 Separation of the CD24 from the IgG1 components ...... 52

2.2.5 CD24 treatment with PNGase F ...... 53

2.2.6 Permethylation of the glycans from CD24 ...... 53

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2.2.7 CD24 deglycosylation with Trifluoromethane sulfonic acid (TFMS) for

glycosylation site determination ...... 54

2.2.8 In-gel digestion of deglycosylated CD24 with protease ...... 55

2.2.9 ESI-MS-MS for the glycans ...... 56

2.2.10 MALDI-ToF-MS ...... 56

2.2.11 LC-MS-MS for peptides ...... 56

2.2.12 Data analysis ...... 57

2.3 Results and discussion ...... 57

2.3.1 Cleavage of the CD24 from CD24IgG1 with papain and its purification ...... 57

2.3.2 Matrix-Assisted Laser Desorption Ionization Time of Flight Mass

Spectrometry (MALDI-ToF-MS) of the released O-glycans ...... 59

2.3.3 Tandem mass spectrometry of the released O-glycans ...... 60

2.3.4 N-glycans ...... 64

2.3.5 CD24 glycosylation site determination ...... 66

2.3.5.1 Deglycosylation of CD24Fc with Trifluoromethanesulfonic acid (TFMSA)

...... 66

2.3.5.2 Digestion of the TFMS deglycosylated protein with proteases ...... 68

2.3.5.2.1 Trypsin ...... 68

2.3.5.2.2 Chymotrypsin ...... 71

2.4 Conclusions...... 73

xiii

References ...... 75

CHAPTER 3 Glycan structure analysis of Fea1 from Chlamydomonas reinhardtii (Algae)

...... 79

3.1 Introduction ...... 79

3.1.1 Fe-assimilating protein 1 (Fea1) ...... 79

3.2 Experimental ...... 80

3.2.1 Materials ...... 80

3.2.2 Glycoprotein (Fea1) detection ...... 80

3.2.3 The release of N-glycans from Fea1 using PNGase A ...... 82

3.2.4 Separation of PNGase A released glycans from peptides with C-18 cartridges

...... 82

3.2.5 Monosaccharide analysis of Fea1 with GC-MS ...... 82

3.2.6 GC-MS analysis ...... 83

3.3 Results and Discussion ...... 84

3.3.1 Glycodetection of the Fea1 from Chlamydomonas reinhardtii results ...... 84

3.3.2 Fea1 monosaccharide analysis with GC-MS ...... 85

3.3.3 MALDI-TOF Mass spectrometry of the released Fea1 Glycans ...... 87

3.3.4 Determination of the glycosylation sites of Fea1 ...... 88

3.3.4.1 Analysis of tryptic peptides with MALDI-TOF-MS ...... 88

3.4 Conclusions ...... 92

xiv

References ...... 94

CHAPTER 4 : Glycosylation analysis of recombinant Tumor Necrosis Factor Receptor.

...... 95

4.1 Introduction ...... 95

4.1.1 Tumor necrosis factor (TNF) ...... 95

4.1.2 Tumor necrosis factor receptor (TNFR) ...... 95

4.2 Experimental ...... 96

4.2.1 PNGase F and PNGase A digestions ...... 96

4.3 Results and Discussion ...... 96

4.4 Conclusions ...... 98

References ...... 100

CHAPTER 5 : Paraoxonase (PON1, the G3C9 Variant) Expression and PEGylation for

Bioscavenger Studies...... 101

5.1 Introduction ...... 101

5.1.1 Paraoxonase ...... 101

5.1.1.1 History...... 101

5.1.1.2 Types of paraoxonases ...... 101

5.1.1.3 Human PON 1 features and structure ...... 102

5.1.1.4 PONs functions ...... 103

5.1.1.5 Paraoxonase in health and diseases ...... 104

5.1.1.6 PON as an OP inactivator ...... 104

5.1.2 Organophosphate Compounds ...... 105

5.1.3 Endotoxin removal from G3C9 and PEGylated G3C9 ...... 108

5.1.3.1 Endotoxins ...... 108

5.1.3.2 Endotoxin removal...... 111

5.1.3.2.0 Methods of removal...... 111

5.1.3.2.1 Ion -exchange chromatography...... 111

5.1.3.2.2 Ultrafiltration;...... 111

5.1.3.2.3 Affinity adsorbents ...... 111

5.1.3.2.4 Two-phase micellar system ...... 112

5.1.3.2.5 Endotoxin removal with Polymyxin B ...... 112

5.1.3.3 Endotoxin assaying ...... 113

5.1.3.3.1 The LAL gel-clot assay ...... 113

5.1.3.3.2 Turbidimetric LAL assay ...... 114

5.1.3.3.3 Chromogenic LAL assay ...... 114

5.2 Experimental ...... 114

5.2.1 Materials ...... 114

5.2.2 G3C9 Transformation and expression ...... 115

5.2.3 G3C9 Purification ...... 115

5.2.4 G3C9 purification using Ni-NTA Column (Trx-His fusions) ...... 116

xvi

5.2.5 PEGylation...... 116

5.2.6 Chromatographic purification of the PEGylated G3C9 ...... 117

5.2.6.1 Size Exclusion Chromatography...... 117

5.2.6.2 Anion Exchange Chromatography ...... 117

5.2.7 G3C9 Enzyme kinetic assays before and after PEGylation ...... 118

5.2.7.1 Determination of the Kinetic constants with the Michaelis-Menten

equation ...... 120

5.2.8 Endotoxin removal and measurements ...... 120

5.2.8.1 Endotoxin removal ...... 120

5.2.8.2 Endotoxin assays ...... 121

5.3 Results ...... 121

5.3.1 G3C9 expression and purification ...... 121

5.3.2 G3C9 PEGylation ...... 122

5.3.3 PEGylated G3C9 purification ...... 123

5.3.3.1 Size exclusion chromatography (SEC) ...... 123

5.3.3.2 Anion Exchange chromatography (AEX) ...... 124

5.3.4 G3C9 and PEG-G3C9 kinetic studies ...... 126

5.3.5 Endotoxin removal and testing of the G3C9 and PEG-G3C9 samples ...... 129

5.4 Conclusions ...... 131

References ...... 132

xvii

CHAPTER 6 Summary ...... 138

Bibliography ...... 142

Appendices ...... 162

Appendix A. O-glycan Chemical Cleavage ...... 162

I. Ammonia-based β-elimination ...... 162

II. β-elimination ...... 162

Appendix B. CD24Fc fragmentation with papain ...... 164

Appendix C. Papain digests purification with protein A ...... 165

Appendix D. Digestion with Enzymes ...... 166

I. Protein digestion with PGase F ...... 166

II. Protein digestion with PGase A ...... 166

III Trypsin in-solution digestion ...... 167

IV. In-gel trypsin digestion ...... 167

Appendix E. Desalting glycans with non-porous graphitized carbon column ...... 169

Appendix F. Permethylation with cartridges...... 170

Appendix G. CD24 deglycosylation with TFMSA ...... 171

Appendix H. GC-MS of alditols acetates ...... 173

Hydrolysis of Glycans from Glycoproteins ...... 173

Reduction of Monosaccharides to Alditols ...... 173

Acetylation of Alditols ...... 174

xviii

Appendix I. G3C9 expression ...... 175

Appendix J. Ni column purification ...... 176

Appendix K. Anion exchange chromatography ...... 177

Appendix L. Kinetic activity Measurements...... 178

Appendix M. Endotoxin removal ...... 179

Appendix N. Figures ...... 180

xix

List of Tables

Table 1.1. Structures of O-antigens ...... 11

Table 2.1. O-glycans released from CD24 by a β-elimination with subsequent permethylation ...... 63

Table 2.2. Permethylated N-glycans from CD24 released by PNGase F and ammonia based beta-elimination method ...... 65

Table 2.3. Summary of CD24 fusion protein tryptic peptides observed after analysis with

MALDI-TOF-MS ...... 70

Table 3.1. Permethylated N-glycans from Fea1 ...... 88

Table 3.2. Identification of Fea1 tryptic peptides after MALDI-TOF mass spectrometry with PeptideMass software ...... 90

Table 5.1. Names and structures of Organophosphate compounds used as nerve agents

...... 107

Table 5.2. Reaction conditions for kinetic measurements for G3C9 using paraoxon .... 119

Table 5.3. UV spectrophotometric data for the appearance of p-nitrophenol from the hydrolysis of paraoxon with G3C9 and calculation or initial velocities ...... 127

Table 5.4. Kinetics reaction results of G3C9 and PEG-G3C9 ...... 129

List of Figures

Figure 1.1. Glycoconjugates in intracellular and extracellular compartments1 ...... 1

Figure 1.2. The basic structure of common classes of glycoconjugates in animal cells ..... 2

Figure 1.3. General schematic overview of N-glycoprotein biosynthesis which occurs in the rough ER and the compartments of the Golgi apparatus ...... 4

Figure 1.4. Types of N-glycans. High-mannose, complex, and hybrid. Each N-glycan contains the common core Manα 1–6(Manα1–3)Manβ 1–4GlcNAcβ 1–4GlcNAcβ -Asn

(Man3GlcNAc2Asn). Glycan structure drawing used from glycoworkbench software tools3, 4 ...... 5

Figure 1.5. Most common O-glycan core structures...... 6

Figure 1.6. The Man 9 glycoform of RNase B. Model based on the 2.5Å X-ray crystal structure16 ...... 8

Figure 1.7. Scheme for sugar cleavage from glycoproteins by hydrazinolysis ...... 16

Figure 1.8. Generalized diagram of an N-linked oligosaccharide attached to an asparagine residue and enzymatic cleavage positions. Additional sugar linkages are possible as shown by 'X' 'Y' and 'Z' ...... 17

Figure 1.9. Reductive amination of glycans with 2-AB ...... 22

Figure 1.10. Serial lectin affinity column chromatography of tritium-labelled oligosaccharide mixture using Concanavalin A (Con A), Phytohaemagglutinin-E 4 (E4-

PHA), Datura stramonium agglutinin (DSA), and Aleuria aurantia lectin (AAL ) ...... 26

xxi

Figure 1.11. PEG synthesis ...... 28

Figure 1.12. First-generation amine reactive PEG derivatives ...... 30

Figure 1.13. NHS esters activated PEG ...... 31

Figure 1.14. Thiol reactive PEGs. (a) PEG maleimide, (b) PEG iodoacetamide, (c) PEG orthopyridyl disulfide and (d) PEG vinyl sulfone...... 32

Figure 1.15. Attachment of poly(ethylene glycol) to oxidized carbohydrates of glycoproteins ...... 33

Figure 1.16. Schematics DNL technology using IMP457, R2b-DDD2, and R2b-457.

Ribbons indicate 40 kDa branchedPEG; red helix, AD2; blue helix, DDD2; SH, free sulfhydryl groups of engineered cysteine residues ...... 34

Blue stain. (b). non-reducing SDS-PAGE after fragmentation with papain and purification with affinity column: line1 MW marker, line 2: the bound portion containing the Fc part of IgG in CD24IgG1, line 3: the CD24, line 4 CD24IgG1...... 59

Figure 2.2. MALDI-ToF mass spectrum for CD24 O-and N-glycans released by the ammonia based beta elimination method ...... 60

Figure 2.3. Nomenclature for the fragmentation of carbohydrates ...... 61

Figure 2.4. (a). Negative ion spectra of O-glycans released from CD24 by β-elimination and (b). the negative scan mode fragmentation spectra of the 675.4 m/z ion. The sample was dissolved in 25 % methanol in 1 % acetic acid ...... 62

xxii

Figure 2.5. MALDI-ToF mass spectrum for the permethylated N-glycans released by

PNGase F from CD24 ...... 64

Figure 2.6. From L-Right: CD24Fc before, CD24Fc after 1, CD24Fc after 2, MW marker

...... 67

Figure 2.7. MALDI-TOF-MS of TMFS treated CD24 fusion protein before reduction with DTT ...... 67

Figure 2.8. MALDI-TOF-MS spectrum TFMSA deglycosylated CD24FC tryptic peptides

...... 69

Figure 2.9. VDGVEVHNAKTKPREEQYNSTY Chymotrypsin digested CD24Fc peptide

MS2 fragmentation spectrum, ...... 71

Figure 2.10. MS2 data of SSETTGTSSNSSQSTSNSGL from chymotrypsin digest of

CD24 fusion protein ...... 73

Figure 3.1. Labeling scheme for glycodetection with UV illumination ...... 81

Figure 3.2. Reaction scheme for sugar hydrolysis and peracetylation ...... 83

Figure 3.3. SDS-PAGE of the unpurified Fea1. It has two bands at ~43 and 38 kDa ...... 84

Figure 3.4. (a) UV photo after transfer to the PVDF membrane; b) UV photo of the SDS-

PAGE gel; c) the photo of the gel. BSA, OV (ovalbuminT (transferrin) MW (molecular weight marker Fv and Fr are the Fea1 protein. R and V is part of the Fea1 protein (Fea1 band is part of the Fea1 protein (Fea1 band is circled) ...... 85

Figure 3.5. GC-MS chromatogram of alditol acetates from sugar standards mixture ...... 86

Figure 3.6. GC-MS chromatogram of monosaccharide alditol acetates hydrolyzed from

Fea1 ...... 86

xxiii

Figure 3.7. MALDI-TOF-MS of the Fea1 permethylated N-glycans released by PNGase

A ...... 87

Figure 3.8. MALDI-TOF-MS of Fea1 tryptic peptides ...... 89

Figure 3.9. Mascot search results of tryptic peptides (top) and chymotryptic peptides

(bottom) of Fea1. In-gel digestions of the sample were carried out for both...... 91

Figure 4.1. MALDI MS spectrum of the permethylated N-glycans released from

TNFR/Fc using PNGase A...... 97

Figure 4.2. .MALDI MS of permethylated glycan form TNFR/FC using PNGase F only

...... 98

Figure 5.1. The structure of paraoxon ...... 101

Figure 5.2. Model of the structure of huPON1 based on the X-ray crystal structure of a chimeric rabbit PON1 variant. Important residues are rendered as sticks and colored for:

Ca2+ binding (red and purple); putative sites of glycosylation (green); cysteines (orange); and putative site of HDL binding (yellow). Blue and purple residues have been found to affect the activity or specificity of PON1 in derivatization, mutational or evolutionary studies. The preponderance of the activity and structural evidence suggests that the active site lies in a pocket just above (and probably including) the Ca2+ binding site in the middle of the β-barrel. The model was generated with SwissModel from PDB entry

1V04, and the figure was generated with PyMOL...... 103

Figure 5.3. Molecular model of the inner and outer membranes of E. coli K-12225

Abbreviation: PPEtn (ethanolamine pyrophosphate); LPS (lipopolysaccharide); Kdo (2- keto-3-deoxyoctonic acid) ...... 109

xxiv

Figure 5.4. Chemical structure of endotoxin from E. coli O86:B4226. (Hep) L-glycerol-

D-manno-heptose; (Gal) galactose; (Glc) glucose; (KDO) 2-keto-3-deoxyoctonic acid;

(NGa) N-acetyl-galactosamine; (NG) N-acetyl-glucosamine ...... 110

Figure 5.5. Polymyxin B structure Polymyxin B1 (R=CH3), Polymyxin B2 (R=H),

DAB=,α’,γ-diaminobutyric acid) ...... 113

Figure 5.6. NHS activated mPEG reaction with G3C9 ...... 117

Figure 5.7. FPLC chromatogram of the protein supernatant purification with Hitrap

Chelating Nickel column and the SDS-PAGE of the collected sample fractions...... 122

Figure 5.8. G3C9 reaction with PEG. left, pH optimization and on the right PEG equivalents optimization. S is pure G3C9 and M is molecular weight marker ...... 123

Figure 5.9. SEC of PEGylated G3C9. The sample was loaded on HiLoad 16/60 Superdex

200 column and eluted with 20 mM Tris buffer pH 8.0 containing 50 mM NaCl, 1 mM

CaCl2 and 0.1 % Tergitol at a Flow rate was 0.5 mL/min ...... 124

Figure 5.10. SDS-PAGE of the SEC peaks 1. MW marker, 2. G3C9, 3. Peak 1, 4. Peak 2,

5. Peak 3 ...... 124

Figure 5.11. Anion exchange chromatography of PEGylated G3C9, separation was carried out as described in the text. The SDS PAGE of each peak is also as shown ...... 125

Figure 5.12. Paraoxon hydrolysis with PON1 ...... 126

Figure 5.13. The Michelis-Menten plot for G3C9 ...... 128

Figure 5.14. Michelis-Menten plot for both G3C9 and PEG-G3C9 and the fitting parameters ...... 128

Figure 5.15. Spectrophotometric data of endotoxin testing and removal from G3C9 sample. The concentration of the unknown is extrapolated from the graph ...... 130

xxv

CHAPTER 1 : Glycan s structure analysis of glycoprotein and protein PEGylation

1.1Glycosylation introduction

Glycosylation is the formation of Glycoconjugates when mono-, oligo- or polysaccharides are attached to proteins or lipids and are called glycoproteins and glycolipids respectively1. Both glycoproteins and glycolipids are located in the extracellular surface of plasma membrane although some are found in the cytoplasm and nucleoplasm2 as shown in Figure 1.1. The common classes of glycans in human cells are shown in Figure 1.2. This study will focus on the Glycosylation of proteins.

Figure 1.1. Glycoconjugates in intracellular and extracellular compartments1

Figure 1.2. The basic structure of common classes of glycoconjugates in animal cells

1.1.1 Protein glycosylation

Glycosylation is a post translational modification (PTM) of proteins that starts in the endoplasmic reticulum and is finalized in the Golgi apparatus for both eukaryotes and prokaryotes. Eukaryotes depend on glycosyltransferases in the ER and Golgi apparatus to give conserved Glycosylation pathways. This leads to recognizable common core structure for N and O- linked sugars linked to asparagine and serine/threonine respectively. Since prokaryotes lack the ER-Golgi assembly, they have a much more diverse structure with some of the monosaccharide residues not found in eukaryotes. In plants and insects, there exist two glycosyltransferases that don’t exist in mammals where

plants have α-1,3 fucosyltransferase and α-1,2 xylosyltransferase while insects we have the α-1,3 fucosyltransferase.

1.1.2 Mammalian N-linked glycoprotein biosynthesis

The mammalian N-linked glycoprotein biosynthesis process involves three parts.

The first cycle called the phosphodolichol cycle occurs in the rough endoplasmic reticulum where a lipid bound oligosaccharide is synthesized. The structure is the same in animals, plants and single-celled eukaryotes. In the synthesis the donor is an oligosaccharide; Glc3Man9GlcNAc2 which is attached to the lipid dolichol via a phosphoryl group. The lipid dolichol is a long (75-95 carbon atoms), highly hydrophobic polyisoprenoid lipid and which can span the membrane 4-5 times.

The process starts in the ER on the cytosolic face in which two GlcNAc and five

Mannose residues are added to the lipid. Then the molecule is flipped to the lumen side where the other remaining monosaccharides which are linked to the dolichol molecule are added one by one after being flipped from the cytosolic face. The lipid linked monosaccharides are synthesized on the cytosolic face of the ER by reaction of the lipid with UDP-Man and UDP-Glc. The formed oligosaccharide serves as the precursor for all the N-linked oligosaccharides to be synthesized.

The second stage of the biosynthesis is the transfer of the oligosaccharide to the polypeptide in one block. The enzyme that catalyzes this transfer of the oligosaccharide to the asparagine on the nascent polypeptide is called oligosaccharyltransferase. This enzyme determines the presence of the amino acid motif Asn-Xxx-Thr/Ser (where Xxx is any amino acid except proline or aspartic acid) on the protein and it is only to this asparagine that the oligosaccharide will be transported to. This process exclusively takes

process in the lumen of the ER and the amino acids must be located appropriately on the three-dimensional structure of the protein.

Figure 1.3. General schematic overview of N-glycoprotein biosynthesis which occurs in the rough ER and the compartments of the Golgi apparatus

The last part of the biosynthesis is the processing of the oligosaccharide; after the transfer, the three terminal glucose residue and a mannose residue are trimmed with glucosidase in the ER. The completion of the trimming of these residues in the ER is the signal for the glycoprotein to be moved to the Golgi. Then the glycoprotein is transferred to the Golgi compartments where further trimming and addition of monosaccharide residues takes place. This process is carried out by glycosidases for trimming and glycosyltransferases for monosaccharide residue addition. Figure 1.3 shows the general overview of the N-glycan biosynthesis.

1.1.2.1 Types of N-linked oligosaccharides

The processing in the Golgi compartment will generally result in oligosaccharides that are heterogeneous in nature. These structures can be bi-, tri- and tetra-antennary with

the bi-antennary being the most abundant. The types of glycans that are generated are high-mannose type with five to nine mannose residues; the hybrid type which has one branch of mannose residues only and the other branch a mixture and the complex type which normally has only three mannose residues with GlcNAc residues attached to the two terminal mannose residues. More monosaccharide residues are normally added to the

GlcNAc residues for further elongation. These types of glycan structures are shown in

Figure 1.4. In general N-linked glycans have a similar pentasaccharide chore with two

GlcNAc and three mannoses.

Figure 1.4. Types of N-glycans. High-mannose, complex, and hybrid. Each N-glycan contains the common core Manα 1–6(Manα1–3)Manβ 1–4GlcNAcβ 1–4GlcNAcβ -Asn (Man3GlcNAc2Asn). Glycan structure drawing used from glycoworkbench software tools3, 4

1.1.3 Biosynthesis of O-linked glycoproteins

This process takes place in the Golgi apparatus in eukaryotes where monosaccharide residues are directly added stepwise to the polypeptide. The first step is

the transfer of N-Acetylgalactosamine residue from UDP-GalNAc to the hydroxyl group of Ser or Thr residue which is catalyzed by N-Acetylgalactosaminyltransferase.

Compared to the N-linked glycans there is no consensus motif for this linkage however, the presence of a proline residue at either -1 or +3 relative to the serine or threonine is favorable for O-linked glycosylation. Then the protein moves to the trans-Golgi vesicles where the carbohydrate chain is elongated in different pathways resulting in different cores. Core 1 structure is generated by the addition of galactose. The core 2 structure is generated by the addition of N-acetyl-glucosamine to the N-acetyl-galactosamine of the

Core 1 structure. Core 3 structures are generated by the addition of a single N-acetylglucosamine to the original N-acetyl-galactosamine. Core 4 structures are generated by the addition of a second N-acetly-glucosamine to the Core 3 structure.

Other core structures are possible, although they are less common. The last step in biosynthesis of typical O-glycans is the additions of N-Acetylneuraminic acid (sialic acid) residues in the trans-Golgi apparatus. The O-glycan structures described above are shown in Figure 1.5.

Figure 1.5. Most common O-glycan core structures

1.1.4 Biological functions of glycoproteins

Glycoproteins are involved in a number of biological functions, these include; a purely structural role, an aid in the conformation and stability of proteins, the provision of target structures for microorganisms, toxins and antibodies, the masking of such target structures for example the 9-OAcetylation of the terminal sialic acid masks the recognition by influenza A and B viruses5, control of the half-life of proteins and cells, the modulation of protein functions, and the provision of ligands for specific binding events mediating protein targeting, cell-matrix interactions or cell-cell interactions.

On the structural function6-8, sugars shield significant areas of proteins from proteases or antibodies because of their relatively large size compared to protein domain

(Figure 1.6) and they also play a role in the spacing and orientation of cell surface proteins. Some oligosaccharides act as recognition epitopes of the proteins that they are attached to because of their particular structure. Examples include the use of the calnexin pathway which is facilitated by monoglucosylated oligomannose sugars9, 10 in the proper folding of proteins and if the protein is not properly folded, it will be degraded, the migration cell surface proteins with sugars carrying the sialyl Lewis X epitope enable cells to sites of inflammation11, and the clearing of proteins with tri-antennary sugars terminating in N-acetylglucosamine by binding to the asialoglycoprotein receptor12.

On the function of being receptors for noxious agents, oligosaccharides on cell surfaces have been shown to act as receptors13 for viruses (eg HIV, influenza) bacteria and parasites and plant and bacterial toxins14, 15. They also serve as antigens for autoimmune reactions.

Figure 1.6. The Man 9 glycoform of RNase B. Model based on the 2.5Å X-ray crystal structure16

Another function is as hormones for example, free oligosaccharides acting as hormones for instance high mannose glycans have high immunosuppressive effects in invitro in mammalian systems6. Glycoproteins that function as hormones include human chorionic gonadotropin (HCG) and erythropoietin which regulates the production of erythrocytes.

Some glycoproteins also serve as transport vehicles after binding to certain molecules for example hormones, vitamins and cations and some decrease the freezing point of sera in some Antarctic fishes due to their interaction with water.

1.1.5 Protein glycosylation and disease

Several studies have been carried out to demonstrate the correlation of glycosylation and the disease although it still remains a challenge. Examples of these diseases are discussed below:

Congenital Disorders of Glycosylation (CDGs): this human genetic disease is quite rare 1/50,000-1/100,000) and has been shown to appear when there is less N- glycosylation due to defects in the assembly of the Dol-P-linked oligosaccharide N- glycan precursor and/or its transfer to the nascent polypeptide17, 18. This is due to the defects in phosphomannomutase, lack of GlcNAc transferase II enzyme which is responsible for the chitobiose core of the N-linked sugars addition, Phosphomannose isomerase deficiency, β-1,4-galactosyltransferase I deficiency, Glucosyltransferase I deficiency, Mannosyltransferase VI deficiency, Dolichol-phosphate-mannose synthase-1 deficiency, GDP-fucose transporter defect, N-acetylglucosaminyltransferase II deficiency,

Mannose-P-dolichol utilization defect, and Glucosidase-I deficiency19. Some of these defects lead to developmental delays, seizures, liver disease, mental retardation, abnormal blood coagulation and muscle loss in the individuals affected.

Bleeding disorder also called von Willebrand disease (VWD) caused by the deficiency of von Willebrand Factor (VWF): VWF is important for the initiation of blood clotting in case of injury. Deficient glycosylation has been found to be one of the reasons for this disorder. For example in one study20, the reduced ST3Gal-IV-mediated sialylation was associated with reduced VWF plasma levels. This means that sialylation is important in preventing immature clearance by binding to receptors like the asialoglycoprotein receptor. In this disease, abnormal expression of N-acetylgalactose

transferase leads to its mislocation to the endothelium instead of the epithelium. The

VWF is normally expressed in the endothelial area and this can lead to its glycosylation by the mislocated transferase leading to the factor being cleared from circulation by binding to the asialoglycoprotein receptor.

Chemical glycation: This process for proteins is possible in diabetic individuals due to excess sugar in the blood. An example is the glycation of the eye lens proteins being involved in the formation of cataracts21, 22. In this process, glucose or other reducing sugars react with the ε-amino group of lysine residues or amino termini of proteins resulting in the formation of schiff base which can undergo further Amadori rearrangement leading to formation of a stable ketoamine23, 24. Glycation of lens protein is believed to enhance their misfolding and thus altering their physicochemical properties as well as their functions.

Rheumatoid arthritis: Studies have clearly shown that there is altered N- glycosylation of serum IgG in rheumatoid arthritis when compared to normal cases25, 26.

In this case it appears that there is an absence of galactose leading to absence of addition of sialic acid27. But it is not clear if lack of the glycosylation is the cause of the disease.

Cancer: Changes in glycosylation are associated with cancer. For example, extensive sialylation of cell surface glycoproteins has been correlated to metastasis potential. This is because sialic acid binding lectins called siglecs especially selectins are known to mediate cell adhesion and extravasation of normal cells. These carbohydrates are called “tumor-associated carbohydrate antigens” (TACA)28. However, changes in glycosylation may be useful in detection and monitoring of cancer. An example is the sialyl-Tn antigen (Neu5Ac 2,6GalNAc-Ser/Thr) which had been found to be presence

in various carcinomas. Others are T, sialyl-T and Tn antigens which mostly found in mucin-type glycoproteins are shown in Table 1.1.

Table 1.1. Structures of O-antigens Name: T Sialyl-T Tn Sialyl-Tn

Antigen

structure :

Ser/Thr Ser/Thr Ser/Thr Ser/Thr

Leukocyte-adhesion deficiency type II (LAD II): This is a rare severe immunological disease which results in severe mental retardation and short stature29. The cause for this disease is due to the absence of sialyl Lewis X on the neutrophils which is a ligand to selectins30.

1.1.6 Therapeutic glycoproteins and carbohydrates

The use of glycoproteins in treatment of diseases and their development is on the rise. Currently, there are several therapeutic glycoproteins in the market for example erythropoietin, granulocyte macrophage-colony-stimulating factor31, and tissue plasminogen activator. The erythropoietin drugs for instance generated about $10 billion dollars in sales worldwide in 200832. These glycoproteins are produced as recombinant proteins and the glycosylation has to be controlled because it contributes to the serum half-life of the glycoprotein, its stability, action and pharmacodynamics on organisms.

For example the glycosylation effect on erythropoietin’s half-life has been studied33. It was noted that low glycosylation led to its quick renal clearance by filtration. It was also

observed that full sialylation of therapeutic glycoproteins glycan chains reduced their clearance by the Gal/GlcNAc/Man receptor.

Carbohydrates are now being used or evaluated for use in blocking the initial attachment of microbes and toxins. This is because some microbes first attach to the cell via some glycans as stated in Section 1.1.4. An example is Zanamivir which is a neuramidase inhibitor used in the treatment of human influenza virus A34. Another example is the polyvalent antigen-KLH anti-cancer vaccine now in clinical trials in which the protein keyhole limpet hemocyanin (KLH) is conjugated to five tumor-associated antigens (TAAs) globo H, GM2 ganglioside, Tn-MUC1, TF, and sTn. This vaccine induces the production of antibodies against tumor-associated antigens35. Another carbohydrate based inhibitor is acarbose which is used in treatment of type 2 diabetes.

Acarbose works by inhibiting the α-glucosidase enzymes that hydrolyze carbohydrates in the brush of small intestines and α-amylase in the pancreas.

Another example of use of glycans in therapy is the use of enzyme replacement therapy targeting lysosomal enzymes for Gaucher’s disease. This disease causes lysosomal storage disorder in which there is an accumulation of the glycolipid glucocerebroside in the macrophages due to the absence of the enzyme glucosylceramidase36. In this therapy, the enzyme, a recombinant N-glycoprotein with terminal mannose residues is produced to target the lysozyme macrophages via the cell surface mannose receptor. This recombinant enzyme is marketed as cerezyme.

1.2 Analytical Methods for Glycoprotein and Glycan Characterization

The challenges for analytical glycobiology is far greater than those in proteomics, however the use of modern separation technologies and new mass spectrometry technologies have contributed a lot in structural elucidation of these structures. For the analysis of glycans from glycoproteins, these sugars structures need to be released before their analysis is carried out. As mentioned earlier, glycan structures are normally complex due to having different linkages, anomericity and due to the fact that they are heterogeneous making it difficult to obtain their complete structures.

1.2.1 Intact glycoproteins analysis

Intact glycoproteins are difficult to analyze because most of them are large in size and some of them maybe in small quantities. Successful methods for analysis of intact glycoprotein include: High performance Capillary electrophoresis (HPCE) which resulted in partial or complete resolution of the glycoforms of several glycoproteins for example ribonuclease B37, bovine fetuin38, horse radish, ovalbumin, recombinant human

39, 40 38, 41 ethythropoietin (EPO) products and 1-acid glycprotein .

Another method that has been used in purification of glycoproteins is lectin affinity chromatography. Some lectins that have high specificities toward sugars are immobilized on matrices and depending on the glycan moieties on the glycoproteins separation is achieved42. The most common lectins that have been used are concavalin A and wheat germ agglutinin. However due to the broad specificities of these lectins, pools of glycoprotein are obtained but not by the structural types.

The other method is mass spectrometry; both matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF-MS) and electrospray mass spectrometry (ESI-MS). MALDI-TOF has been shown to resolve glycoforms in a case where there the biomolecule was small and only had one glycosylation site43. For large biomolecules generally large unresolved peaks are observed44. In ESI-MS, only using nano-ESI-MS has shown some success in resolving

45 some glycoproteins an example being the 1-acid glycoprotein . In general desolvation in ESI is less effective for glycoproteins due to the attached oligosaccharides. To obtain complete structures of the attached glycans which includes linkage, branching, composition and conformation, the glycans will need to be released and analyzed independently or digested with proteases and the glycopeptides analyzed. The glycan release/analysis and glycopeptide analysis is discussed in the sections that follow.

1.2.2 Glycopeptide analysis and site-specific glycosylation analysis

This is normally carried out to determine the glycosylation sites and also determine the glycan heterogeneity on each site. In the analysis normally the glycoprotein is digested with trypsin or any other suitable endoprotease. Then the glycopeptides are isolated from the peptides for MS study since the peptides suppress their ionization. In one study, after protease digestion of glycoprotein they analyzed the digests directly by

MALDI-MS, another portion after N-glycan cleavage and the last portion after O-glycan removal and were able to determine the O- and N-glycosylation sites46. Other N- glycosylation sites determination methods have been carried out with MALDI-MS include; for -human interferon- by use of immobilized trypsin cartridge and monitoring

for glycopeptides shifts47. Another method involved enzymatic N-glycan removal with

18O-labeling of glycosylated asparagine48.

In another study the glycopeptides were generated by enzymatic digestion with pronase followed by analysis with MALDI-FT-MS49. Also MALDI-TOF-TOF was used to provide the N-glycosylation sites, peptide sequence and glycan structure of horseradish peroxide50. These methods of digestion have also been used in studies that the digests were analyzed by HPLC coupled with ESI-MS. This allowed for separation of the peptides generated and detection with MS-MS51-53. Examples in these studies include the glycopeptide mapping of rt-PA54, complement receptor I55, bovine fetuin56 and recombinant human thrombomodulin57.

Other mass spectrometric methods for site determination that have been used are

Fourier transform ion cyclotron resonance MS58 (FTICR-MS) which has been used by fragmenting the glycopeptides with collision activated dissociation (CAD)59, infrared multiphoton dissociation (IRMPD) and electron-capture dissociation (ECD)59-61. ESI coupled with capillary electrophoresis (CE) to provide faster and higher separation efficiency than HPLC has also been used in glycopeptide analysis with high sensitivity62-

64.

1.2.3 Cleavage of glycans form glycoproteins: chemical and enzymatic

1.2.3.1 Chemical cleavage

1.2.3.1.1. Hydrazinolysis:

N- and O-glycans can be released by careful treatment of the glycoprotein with anhydrous hydrazine. In this method, care should be taken at all times to ensure there is

absolutely no water in the reaction mixture. The reaction is carried out by the addition of hydrazine to an appropriate amount of glycoprotein in an air tight tube and the reaction performed for four to six hours in an oil bath at 60 °C for and 95 °C for N-glycans65, 66.

The reaction scheme is shown below.

STEP1 HOH C HOH2C H 2 O H H2 Hydrazinolysis O O r S O N N e-N TE C NH R HO NH2 -a P R HO C ce 2 NH C C O NH2NH2 NH2 ty H lat (hydrazine) A ion C O O acetohydrazone c O H C 2 3 HOH C cleavage O 2 O HOH2C H O OH O O N C R HO Mild acid hydrolysis R HO N NH CH3 STEP 3 NH2 H Free-reducing end glycan C O H3C

Figure 1.7. Scheme for sugar cleavage from glycoproteins by hydrazinolysis

However this method has largely been replaced by enzymatic release because it suffers several problems like peeling of the reducing terminal GlcNAc because of high temperatures and the need for re-N-acetylation step which for sialic acid might result in a different type of sialic acid.

1.2.3.1.2. Alkaline β-elimination

This method67 only releases O-glycans as alditols due to the reduction step to minimize peeling reactions caused by the basic conditions. In this method, a sample of freeze dried glycoprotein is dissolved in NaOH containing sodium borohydride and incubated at 45 °C. The mixture is then desalted by cation-exchange before further analysis. But due to formation of alditols, reductive amination is not possible for attachment of fluorophores for detection by UV. Even for MS detection68, it suffers low

sensitivity due to the poor ionization efficiency of glycans. Another β-elimination method is the so-called Ammonia-based β elimination69: this method releases both N and O- glycans although the N-glycans are not released quantitatively. This method is good for

MS since the reducing end is left intact due to the mild conditions of the reaction.

1.2.3.2 Enzymatic cleavage

The most effective method for the release of N-glycans from glycoproteins is the use of PNGase F which is available commercially. The enzyme actually cleaves the bond between the sugar and asparagine converting it to aspartic acid. It however does not cleave glycans that have α-1,3 linkage to the reducing end GlcNAc. These type of glycans found mostly in plants can be cleaved by PNGase A 70. Endoglycosidases have also been used but they cleave the bond between the two GlcNac of the chitobiose core leaving one GlcNac on the protein. An example is endoglycosidase H (Endo H) which has been used to cleave both the hybrid and himannose glycans. The general enzymatic cleavage of N-glycans is shown in Figure 1.8.

Z X Y Asn X Endoglycosidase Glycoamidasidase

Figure 1.8. Generalized diagram of an N-linked oligosaccharide attached to an asparagine residue and enzymatic cleavage positions. Additional sugar linkages are possible as shown by 'X' 'Y' and 'Z'

For O-glycans, there is no universal reliable enzyme for the cleavage. The only available enzyme is the endo--acetylgalactoamidase which cleaves the unsubstituted core-1 O-glycans.71

1.2.4 Analysis of the released glycan mixtures.

1.2.4.1 Mass Spectrometry

The advance in the development of mass spectrometry (MS) in the recent years has had a lot of impact in the analysis of glycans and glycoconjugates. This is because mass spectrometry has many advantages over the traditional analytical methods in that it has high sensitivity and requires low amounts of sample. MS is more robust when coupled with modern separation methods in carbohydrate structural elucidation. The MS method originally used for carbohydrate analysis was Fast atom Bombardment (FAB-

MS)72 which provided molecular weight, linkage and sequence information but has been gradually replaced by softer ionization methods like electrospray (ESI-MS) and matrix assisted laser desorption ionization (MALDI-MS)73-76.

1.2.4.1.1 FAB-MS

Fast atom bombardment mass spectrometry was first introduced in the early

1980s77, 78. In this method the sample must be dissolved in a suitable non-volatile matrix like glycerol. FAB-MS is a useful method for the analysis of oligosaccharides and provides useful information for example branching and sequence. In FAB, a sample that has been dissolved in a suitable matrix is bombarded with 8-15 keV Cs+ ions. Following ionization, the selected positive or negative ions are extracted, accelerated, and their mass

analyzed. For better ionization of glycans, permethylation is performed to provide better sensitivity and characteristic maps of the fragmented ions79.

This method has been used to obtain glycan profiles of murine organs for example tissue and in the process help in revealing their roles in disease80. Other studies have determined the structures of glycans released from Tamm-Horsfall a glycoprotein associated with human urine81, megalin, a cell surface receptor82, and glycoproteins from egg white of pigeons83. This method however has several disadvantages like a big drop in sensitivity at higher molecular weight when compared to ESI or MALDI, high background in the MW range of 200-500 Da and requirement of solubility of the sample in the matrix. This has led to this method being replaced by ESI and MALDI in the analysis of oligosaccharides.

1.2.4.1.2 ESI-MS

Electrospray MS is a soft ionization method that has become an important technique in the analysis of carbohydrates and is widely used. It provides efficient ionization and multiply charged ions which are important factors for large biomolecules analysis. N-glycans analysis with ESI-MS was first reported by Duffin et al.84 in which they studied underivatized sialylated glycans in the negative mode and neutral ones in the positive. Since then ESI has been used to study all types of eukaryote and prokaryote glycans. The use of tandem ESI-MS (MS-MS) has been developed to fragment oligosaccharides and provides both composition and sequence information. The ions are fragmented by collision induced dissociation (CID) which is a method by which a precursor ion is introduced in a collision cell containing a high pressure of an energized,

chemically inert collision gas (Ar, He, N2, CO2). The fragmentation occurs due to the repeated collision with the collision gas. Most of these studies have been done where the

ESI-MS is coupled with LC for separation because glycans released from glycoproteins are heterogeneous mixtures. For better sensitivity, and analysis of sub picomole amounts, the glycans were derivatized and analyzed with a nano ESI-MS85-89.

Other ESI methods that have been used in glycan analysis are ESI-IT (ion trap)90-

92; ESI-QTOF with nanospray which was used to analyze derivatized himannose, hybrid and complex type of N-glycans93, underivatized N-glycans94, sialylated N-glycans95, sulfated oligosaccharides from mucins96, 97 and underivatized O-glycans. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used with infrared multiphoton dissociation (IRMPD) for the analysis of glycoproteins from 2D gels. This method is very sensitive and provides high accuracy and with use of IRMPD deglycosylation was not required since the dissociation occurs at the peptide glycosidic bonds98, 99.

1.2.4.1.3 MALDI-MS

This method, developed in the late eighties has advantages over FAB-MS in that it has 10-100 fold higher sensitivity100, lower suppression101 and can analyze higher molecular weight glycans. Compared to ESI, MALDI-MS has a higher tolerance of salts and buffers102, 103. Also the ions generated are normally singly charged therefore easier to analyze. This technique has been widely used in the analysis of glycan mixtures from glycoproteins especially N-glycans. One of the first reports of oligosaccharide analysis with MALDI-TOF-MS was in which they analyzed α-glucan, maltoheptaose and

cycloheptaose43, 104.

In other studies105-107, an array of exoglycosidases was utilized to obtain the complete structure of underivatized glycans by carrying out enzyme digestions directly on the MALDI target. MALDI-TOF-MS has been used in glycan profiling of sialylated

N-linked glycans108, reports of studies on fragmentation have been studied for example

PSD fragmentation of oligosaccharides by Yamagaki and co workers109, MALDI/CID spectra of milk sugars and N-glycans with external source FT-MS110, and complex N- glycans analysis with MALDI quadrupole/time of flight MS93. MALDI TOF-TOF-MS has also been used to sequence glycans utilizing its high resolution for example the sequencing of glycans from CON-S gp140ΔCFI protein111.

1.2.4.2 Chromatography

1.2.4.2.1 Use of reverse-phase (RP) columns

The commonly used column is the C-18 RP-LC. However, oligosaccharides are generally poorly retained on this column. In general using water as a mobile phase, more polar oligosaccharides are eluted before the less polar ones and retention increases with molecular weight mass. For efficient separations, the sugars are generally derivatized with hydrophobic tags to increase retention and also provide fluorophores for detection.

For this purpose several tags have been used, Hase and co-workers112 derivatized several glycans from various proteins with aminopyridine (2-AP) by reductive amination followed by analysis with RP-LC. The sample was cleaved from a protein sample as little as 0.15nmol. Labeling the glycans with 2-AP tag, separation with two dimensional LC was carried out with RP in one dimension and normal phase in the other by Tomiya et

al.113. In this work a database of 113 standards was created by comparing unknowns to standards and 1HNMR spectroscopy. Another derivatization agent is the 2- aminobenzamide (2-AB)16, 114, 115. The general scheme for reductive amination with 2-AB is shown in Figure 1.9.

HOH2C HOH C HOH2C OH HOH2C 2-AB 2 OH Reduction RO O OHO N RO HN RO RO NH HO OH HO HO NH2 HO 2 NHAc AcHN H AcHN H AcHN H O O

Figure 1.9. Reductive amination of glycans with 2-AB

Depending on the sugar linkages, the 2-AB labeled sugars were compared to glucose units (GU) generated from the dextran ladder. These were then compared to standards which were used to create a database for glycan linkage analysis114.

Other labeling compounds have been used followed with RP and LC MS include

1-phenyl-3-methyl-5-pyrasolone (PMP)116, 117, phenylhydrazine118, 2-aminoacridone (2-

AMAC)119, 120, 3-(acetylamino)-6-aminoacridine (AA-Ac) for both RP and normal phase121 and 2-amino-5-bromopyridine which allow straight forward differentiation of the Y-type and the B-type fragments85. Another approach is permethylation of the sugars which has been used by different groups92, 122, 123. Permethylated glycans are sensitive to

ESI-MS detection and provide a lot of linkage information in MS2 and MS3.

1.2.4.2.2 Use of normal-phase (NP) columns

Normal phase HPLC also called hydrophilic interaction chromatography has been used in separation of glycans labeled by mainly 2-AP and 2-AB. The labeled

oligosaccharides are retained by hydrophilic interactions and eluted by increasing the aqueous buffer concentrations. The stationary phase used is normally silica-based terminating with amide or amino. 2-AB labeled glycans that have been released from ribonuclease B, 1-acid glycoprotein have been separated on this type of column and the separation was not only according to size but also branching124. In another study, a reproducible method was devised in which the elution positions of the standard glycans labeled with 2-AB were determined in glucose units (GU) in reference to the dextran ladder125. This permits the identification of oligosaccharides including their linkages according to the GU value obtained. The use of unlabeled glycans like O-alditols has been used with NP-nano-LC-MS with sensitivity of up to 1 fmol.126

1.2.4.2.3 Use of Graphitized carbon columns

Graphitized carbon columns (GCC) were introduced later127, 128 after NP and RP for oligosaccharide analysis. The oligosaccharides are retained primarily by adsorption and hydrophobic interactions and generally eluted with increasing the amount of acetonitrile in trifluoacetic acid. This material is unique in selectivity in that it can resolve isomeric127 and compounds that are closely related. These columns are very stable and can be used at a wide pH range. The first studies on GCC were analysis of disaccharide and cyclomaltoses127 then followed by bovine fetuin N-glycans with offline LC-MS detection128. The separation was based on size, charge and linkage. Several other studies on the analysis of glycans detected by online LC-MS have been carried out. The first study was analysis of neutral and acidic oligosaccharides by Kawasaki et al.129 They also were able to carry out the sugar analysis released from 50 ng of erythropoietin. In another

study, GCC was able to separate pronase released glycans from soybean agglutinin in 30 min, 10-30 % aqueous acetonitrile while C-18 did not retain the glycans even when water was used to elute.130 Coupling GCC and HILIC with ESI-MS was found to permit complete characterization of sulfated mucin oligosaccharides since each column had different selectivity and good resolution of the mixtures131.

1.2.4.2.4 High-Performance Anion-Exchange Chromatography (HPAEC)

HPAEC coupled to pulsed amperometric detector (PAD) is a method that has been in use for carbohydrate analysis because of its analysis speed, fairly high sensitivity without derivatization and adequate separation of anomeric structures. The method is based on the anionic interaction between the anion exchange resin and the negatively charged oxyanions that result from the use of highly basic mobile phase (pH>12). In general the negatively charged oligosaccharides are eluted last after the neutral carbohydrates. This method was first used Townsend etal. in which they were able to separate neutral oligosaccharides according to their molecular weight, sequence type and linkage of the monosaccharides. Another group used HPAEC-PAD to elucidate the oligosaccharides structures of human tissue plasminogen activator expressed in Chinese hamster ovary132. This was done in conjunction with 1HNMR, endoglycosidases, other

HPLC methods and FAB-MS.

1.2.4.2.5 Use of Lectin Affinity columns

The use of lectin immobilized columns as described in section 1.2.1 is an effective tool for fractionation of complex mixture of glycoproteins. It is the same case to

complex mixtures of glycans when packed columns with different carbohydrate-binding selectivity are used in series to obtain fractions of radiolabelled oligosaccharides that have different structures133 (see Figure 1.10). And more structural information can be obtained when combined with other analytical methods according to different studies. For example lectin affinity chromatography was combined with Bio-Gel P-4 column chromatography to obtain the structures of oligosaccharides obtained from the lectin of the common bean Phaseolus vulgaris142.

Figure 1.10. Serial lectin affinity column chromatography of tritium-labelled oligosaccharide mixture using Concanavalin A (Con A), Phytohaemagglutinin-E 4 (E4- PHA), Datura stramonium agglutinin (DSA), and Aleuria aurantia lectin (AAL )

1.2.4.2.6 Capillary Electrophoresis

Methods of electromigration that have been used in analysis of oligosaccharides are capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). However these methods require the glycans to be derivatized with a fluorescent tag that is ionic since carbohydrates are generally neutral and have low detection sensitivity to UV or fluorescent detectors. Examples of

derivatizing agents include; 2-aminopyridine, 6-aminoquinoline, 8-aminonaphthalene-

1,3,6-trisulfonic acid and several others are listed on this review135.

CZE has been used in conjunction with UV and Laser Induced Fluorescence (LIF) to successfully analyze glycans from ovalbumin136, ribonuclease B137, bovine fetuin138, 139,

136 139 human IgG , and, 1-acid glycoprotein . This method provides high resolution and depending on derivatization low quantities of sample could be detected with high sensitivity137. In MEKC, the advantage is that neutral derivatizing agent for example 2-

AB and 2-AMAC can be used. The method has been used to separate oligosaccharide

140 141 140, 141 released from ovalbumin , 1-acid glycoprotein , ribonuclease B , bovine fetuin140, and, human IgG142.

CEC is another separation technique that utilizes both best features of CE and

HPLC but is not widely used. Novotny and coworkers demonstrated the effectiveness of

CEC in the separation of ribonuclease B glycans with MS detection in which it allows for high separation efficiency69.

1.2.4.2.7 Fluorophore-Assisted carbohydrate electrophoresis (FACE)

This method was introduced by Jackson in the early nineties and it utilizes polyacrylamide gel electrophoresis (PAGE) for the carbohydrate separations and detection by fluorescence or UV illumination143. The method is able to resolve both mono- and oligosaccharides. The oligosaccharides are generally labeled with 8- aminonaphthalene-1,3,6-trisulfonic acid (ANTS) to confer fluorescence and charge for neutral glycans141. It has enabled the separation of glycans released from glycoprotein like recombinant iduronase144, soluble human interferon receptor 2145 and candida

mannoprotein146. In these studies the glycan mobilities were compared to those of a maltose ladder of 2 to 20 sugar units labeled with ANTS.

Further structural elucidation of an identified glycan was confirmed by use of specific exoglycosidases or as in another study by elution of the glycans from the gel bands followed by analysis with MS147. A new method that has been used in carbohydrate analysis is the DNA Sequencer-Aided-FACE (DSA-FACE) which was used in glycan profiling and disease diagnosis for example liver disease148.

1.3 Protein modification to mimic glycosylation by PEGylation

1.3.1 PEGylation introduction

In cases where the protein expression system does not result in glycosylation for example bacterial, the protein is chemically modified with PEG which mimics glycosylation. PEGylation is the process in which poly(ethylene glycol) (PEG) polymer chains is covalently attached to another molecule, normally a drug or therapeutic protein.

In its common form, PEG is a linear molecule with the formula HO-(CH2CH2-O)n-

CH2CH2- OH. It is synthesized by ring opening polymerization of ethylene oxide initiated by a nucleophilic attack of a hydroxide ion on the epoxide ring (Figure 1.11).

The most common form of PEG available commercially is the methoxy-PEG with general structure: CH3O-(CH2CH2-O)n-CH2CH2- OH.

anionic n HO H Polyethylene glycol O polymerization O n

MeO H Figure 1.11. PEG synthesis Methoxy polyethylene glycol O n

O PEG structure O n

1.3.2 Importance of PEGylation of therapeutic proteins

PEGylation is used to improve pharmacokinetic and pharmacodynamic properties of therapeutic proteins by: shielding antigenic epitopes of the polypeptide thus reducing reticuloendothelial clearance and recognition by the immune system, increasing the apparent size of the polypeptide thus reducing renal filtration, providing water solubility to hydrophobic drugs and proteins, increasing its half-life, increasing its shelf life, decreasing its degradation, and decreasing its proteolysis. There are currently several

FDA-approved PEGylated proteins on the market (Neulasta, Oncaspar, PegIntron, Doxil,

Cimzia, Macugen, and Pegasys), with many others still in clinical development149, 150.

1.3.3 Chemistry of PEGylation

To couple PEG to molecules like proteins or peptides, the PEG needs to be functionalized at one or both ends so that it is reactive towards a functional group on a particular amino acid on a protein. These amino acids include the lysines, cysteines, histidine, arginines, aspartic acids, glutamic acids, serine, threonines and tyrosine. The C and N-terminal are also reactive towards activated PEG149. In case of glycoproteins, the vicinal OHs can be oxidized to form two reactive formyl moieties and be used for

PEGylation. The PEGylation chemistries are outlined below.

1.3.3.1 PEG chemistry for conjugation to amine

First-generation PEG derivatives include: (a) PEG dichlorotriazine151, (b) PEG tresylate, (c) PEG succinimidyl carbonate152, (d) PEG benzotriazole carbonate, (e) PEG p-nitrophenylcarbonate, (f) PEG trichlorophenyl carbonate153, (g) PEG

carbonylimidazole154 and (h) PEG succinimidyl succinate155 the reaction scheme is shown in Figure 1.12. They are involved in the PEGylation of α or ε amino groups.

Cl Cl N R-NH2 N a) PEG O N PEG O N N N Cl HNR

R-NH2 O2 b) PEG O S CH2CF3 PEG NHR O O O R-NH2 c) PEG O C O N PEG O C NH R

O O N O N R-NH2 d) PEG O C O N PEG O C NH R

O O R-NH2 e) PEG O C O NO2 PEG O C NH R

Cl O O R-NH2 f) PEG O C O Cl PEG O C NH R

Cl O N O R-NH2 g) PEG O C O N PEG O C NH R O O O O O R-NH2 h) PEG O C CH2CH2 C O N PEG O C CH2CH2 C O NH R

Figure 1.12. First-generation amine reactive PEG derivatives

Second-generation PEGylation chemistry for amines: These have been designed to avoid diol contamination, low molecular weight mPEG problems, selectivity, unstable linkages and side reactions associated with the First-generation ones156, 157. The general

reaction scheme for second generation PEGylation chemistry is shown in Figure 1.13.

They have NHS esters on based on propionic and butanoic acids with linear and branched

PEG NHS esters.

O O O mPEG O(CH2)nC N + R-NH2 mPEG O(CH2)nCNH R O

O O O O(CH ) HC C NH R mPEG O(CH2)mHC C N + R-NH2 mPEG 2 m Y Y O

Figure 1.13. NHS esters activated PEG

1.3.3.2 PEG chemistry for cysteine modification

This is the most common approach for site-specific PEGylation due to the fact that the number of cysteines that are free in proteins is way less than lysines and hence will mostly result in one modification. PEG derivatives for cysteine residues are shown in

Figure 1.14 158-161.

O O O O SH-R SR a) mPEG O C O N mPEG O C O N

O O O O SH-R b) mPEG NHCCH2I mPEG NHCCH2S R

SH-R c) mPEG S S mPEG S S R N

O O SH-R d) mPEG S CH CH2 mPEG S CH2 CH2SR O O

Figure 1.14. Thiol reactive PEGs. (a) PEG maleimide, (b) PEG iodoacetamide, (c) PEG orthopyridyl disulfide and (d) PEG vinyl sulfone.

1.3.3.3 PEG chemistry for oxidized carbohydrates or N-terminus

This method is an alternative for site-specific PEGylation. In this method carbohydrates are oxidized with either glucose oxidase or sodium periodate to aldehydes162. Another method is by sodium periodate oxidation of the N-terminus if it is a serine or threonine resulting in a glycoxylyl derivative that can be conjugated to hydrazide-PEG.163The reaction scheme is shown in Figure 1.15.

O H Glycoprotein O HO CHOH CHO Activated Glycoprotein

PEG-Hydrazide H Activated Glycoprotein Glycoprotein C (N NHCOCH2-PEG)

PEG-amine Glycoprotein Activated Glycoprotein CH2 (NH PEG) NaCNBH3

Figure 1.15. Attachment of poly(ethylene glycol) to oxidized carbohydrates of glycoproteins

1.3.3.4 Heterobifunctional PEG chemistry

This is where PEG is functionalized with two dissimilar groups.

Heterobifunctional PEGS have a general structure, X-PEG-Y where Y and X are two different functional groups. These are useful in crosslinking or spacing two entities such as linking macromolecules to surfaces. Examples of these types of PEGs include functionalizing with hydroxyl and amino groups164, amino group and NHS, and formyl group on one and hydroxyl group on the other165.

1.3.3.5 PEGylation using dock and lock (DNL) technology

This is a method that exploits specific protein-protein interaction between a protein and its anchoring domain protein (AD). An example of this protein is cAMP dependent protein kinase (PKA) and A-kinase achoring proteins (AKAPs)166. The AD of

AKAPS binds to the PKA subunits RI and RII. The PKA domain is called Dimerization

Docking Domain (DDD) because the R unit dimerizes. The AD will bind with the DDD and can be stabilized further by reaction to covalently form disulfide bridges. Chang and

coworkers167 exploited this phenomenon by binding a the dimer of a fused DDD domain with human interferon-R2b (IFN-R2b) and the AD derivatized with PEG as shown in

Figure 1.16. This resulted in site-specific PEGylation with the units retaining antitumor activity and improved pharmacokinetic properties.

Figure 1.16. Schematics DNL technology using IMP457, R2b-DDD2, and R2b-457. Ribbons indicate 40 kDa branchedPEG; red helix, AD2; blue helix, DDD2; SH, free sulfhydryl groups of engineered cysteine residues

1.4 Research goals

In this research we used MALDI-TOF-MS and LC-MS-MS to obtain the molecular structures of glycans from proteins expressed in different systems. The first analysis is described in Chapter 2 where our goel was to elucidate the glycosylation structures and determine the glycosylation sites of recombinant human CD24Fc glycoprotein. This glycoprotein was expressed in a mammalian system, Chinese hamster ovary (CHO).

The analysis of CD24 is difficult due to its heavy glycosylation and aminoacid sequence lacking proteolytic sites. In the research a method by chemical glycosylation were optimized and used with proteolysis and mass spectrometry with the aim of identifying the glycosylation sites on CD24.

In Chapter 3, a study was carried out to determine what kind of glycosylation is present in Fea1. This is a periplasmic protein from Chlamodomonas reinhardtii a type of algae which is a one cell organism. The glycosylation of algae is thought to be plant-like because of the common ancestry. Therefore, it can be used to express therapeutic proteins for example PON1 which was used in this study and possibly resulting in glycosylation.

These studies to obtain the glycoforms were carried out using GC-MS, MALDI-TOF-MS and LC-MS-MS.

In another study described in Chapter four a glycosylation analysis by MALDI-

TOF-MS was carried out to determine the glycan structures on tumor necrosis factor receptor (TNFR) expressed in Camelina sativa another expression system. Camelina sativa is a plant and has some advantages over mammalian systems in therapeutic protein

expression. The aim was to find if this plant is suitable for therapeutic protein expression in regard to the glycoforms that the expressed protein will have.

Chapter 5 describes PEGylation of a potential bioscavenger. For therapeutic proteins expressed in bacterial system like E. coli, which generally does not glycosylate proteins, it is important to find a cheap alternative that will mimic glycosylation of the therapeutic protein This alternative is normally PEGylation. Therefore our goal here was to express G3C9 a paraoxonase 1 enzyme (PON1) in E. coli and PEGylate it. And also study the effect of PEGylation on the catalytic efficiency of PON1 comparison to the unPEGylated form by performing kinetic studies.

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CHAPTER 2 The glycosylation analysis of recombinant CD24

2.1 CD24 protein introduction

CD24 is a small mucin-like glycosylphosphatidylinositol (GPI)-linked cell surface protein that is expressed in developing or regenerating tissue and also in granulocytes, pre-B-cells, keratinocytes, and renal tubular epithelial cells1-3. It is also expressed in hematological malignancies, and a variety of solid tumors such as gastric, renal, nasopharyngeal, hepatocellular, colonic, and small cell lung carcinoma 4, 5. The protein is composed of 27-35 amino acids and nearly half of the amino acids are serine and threonine. Both serine and threonine are potential sites for O-glycosylation1 and it is known to be N-glycosylated6.

The CD24 glycoprotein plays important roles in both normal cells and cancerous cells. For example, it has been found to be a costimulatory molecule for T-cell activation essential for function of T-cells7. It also is a signal-transducing molecule on the surfaces of most human B cells that “modulate[s] the cell response to activation signals by antagonizing interleukin-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by [antigen] receptors” 8. Concerning cancerous cells, CD24 has been found to be prevalent on many tumors and carcinomas9.

Carbohydrates of CD24 on tumor cells have been shown to play an important role in metastasis by interaction with P-selectin. The interaction of P-selectin and sialylLewisX carbohydrates is essential for CD24-mediated rolling of tumor cells. Adhesion of cancer

cells and the vascular endothelial cells is necessary for the cancer cells to reach outside of the vascular system and facilitate metastasis 3.

CD24 expression is a prognostic marker in breast cancer, and it is believed that the structural characteristics of the glycans found on the protein may predict the course of the disease in the patient, and thus allow for individual-specific treatment 3. In addition to this, it is believed that the carbohydrates on CD24 may facilitate the spread of cancer.

Thus, an understanding of the structure and function of these epitopes will greatly facilitate our understanding of the role of CD24 in cancer3, 9.

Comparative analysis between N-glycans of CD24 protein isolated from mouse brain and human lymphoblastoma, neuroblastoma and astrocystoma cell lines has been carried out6. The results showed that CD24 is highly N-glycosylated with fucosylation and sialylation with the human having higher branching with some similarities and a few differences.

The analysis of the CD24 glycosylation sites is challenge in that this protein has just 30 amino acids and is covered with O-glycosylation which consists of up to 90 % of its molecular weight. This makes it difficult to digest the protein with proteases. Due to lack of an enzyme that can universally remove all O-linked glycans we are left with chemical methods to remove the sugars.

The method of chemical deglycosylation that leaves an intact peptide is by use of

Trifluoromethanesufonic acid (TFMS) which removes all the sugars but leaves the

GlcNAc or GalNAc attached to the peptide. Therefore this method will be used together with enzyme digestion and mass spectrometry to identify the glycans on CD24 and the sites of glycosylation.

2.2 Experimental

2.2.1 Materials

The human CD24IgG1 protein, comprising the first 30 amino acid of human

CD24 protein (SETTTGTSSNSSQSTSNSGLAPNPTNATTK) and the IgG1 Fc, was produced in Chinese hamster ovary (CHO) cells and purified by ion-exchange and protein A-based affinity chromatography to homogeneity. All chemicals and reagents unless otherwise stated were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2, 5 dihydroxybenzoic acid and acetic acid were purchased from Acros Organics (New Jersey,

USA). HPLC grade water, acetonitrile, methanol and n-propanol were purchased from

Burdick and Johnson (Muskegon, MI, USA).

2.2.2 Chemical cleavage by beta-elimination

Since the Fc of IgG contains no O-linked oligosaccharides 10, the CD24IgG1 was freeze dried and the O-glycans released by the ammonia based beta-elimination method as described in Appendix A.I introduced by Huang et al. 11. In this study, ice-cold ethanol was added to precipitate the peptides after boric acid removal. Centrifugation was carried out and the supernatant which contained the sugars decanted for further analysis.

The O-glycans that were analyzed by tandem mass spectrometry were released by the beta-elimination method according to Kotani and co-workers 12 (Appendix A.II).

2.2.3 Cleavage of CD24 from CD24IgG1 with papain

IgG is known to contain N-glycans we therefore cleaved the Fc fragment off in order to focus on the N-glycans from the CD24 glycoproteinusing method describe in

Appendix B. A 20 mg/mL solution of CD24IgG1 in PBS buffer was prepared followed by addition of 0.1 mg/mL papain in digestion solution 13. This mixture was incubated for

5 h in a circulating water bath set at 37 °C. The reaction was quenched by the addition of

400 μL 0.3 M iodoacetamide. This solution (3 mL) was dialyzed overnight at 4 °C against a 3500 MWCO dialysis membrane and freeze dried.

2.2.4 Separation of the CD24 from the IgG1 components

The separation of the CD24 from the fragmented CD24IgG1 was carried out by using a Protein A-Sepharose from Staphylococcus aureus affinity column based on the manufacturer’s protocol (Appendix C). The column was prepared by mixing 0.5 g of

Protein A-Sepharose with 2.5 mL buffer A (prepared by adjusting 0.02 M NaH2 PO4 +

0.15 M NaCl to pH 8.0) and allowing to stand for about 45 min to swell. The swollen resin was mixed with buffer A (1:1, v/v) and poured into the column. The solvent was allowed to drain while the resin settled. This was later washed with 20 column volumes

(CVs) of buffer A. After introducing the sample into the column, 10 CVs of buffer A was used to elute the CD24 components from the column. The Fc component and the unfragmented CD24IgG1 was eluted with 3 CVs of buffer B (prepared by mixing 25.7 mL 0.2M Na2H PO4, 24.3 mL 0.1 M citric acid and 50.0 mL deionized water). SDS-

PAGE was used to test the purity of collected fractions and compared with the unfragmented CD24IgG1 and the fragmented CD24IgG1.

2.2.5 CD24 treatment with PNGase F

CD24 was treated with PNGase F (Sigma-Aldrich) according to Appendix D. In the procedure, 1.0 mg of the CD24 obtained after papain cleavage and purification was dissolved in 900 µL of 20 mM ammonium bicarbonate pH 8.0 and to this solution 100

µL of denaturalization solution was added (0.2 % SDS containing 100 mM 2- mercaptoethanol). The solution was placed for 10 minutes in boiling water to denature the glycoprotein. The solution was allowed to cool and 10 U of PNGase F added to this reaction mixture and incubated at 37 °C for 20 h. Then 10 µL was taken after the incubation to monitor the cleavage of the N-glycans with SDS-PAGE.

The reaction mixture was first freeze dried and then desalted and deproteinized using a non-porous graphitized carbon column as described in Appendix E (Alltech,

Deerfield, IL, USA) 14. The carbograph column was washed with three CVs of 80 % acetonitrile in water (v/v) with 0.1 % (v/v) trifluoroacetic acid, followed by three CVs of de-ionized water. The sample was dissolved in a small amount of water and introduced into the column. The salts and buffer were eluted with 2 mL of ultra-pure water and neutral N-glycans were recovered with 2 mL of 25 % (v/v) acetonitrile in ultrapure water.

Acidic N-glycans were eluted with 2 mL of 25 % (v/v) acetonitrile in water with 0.05 %

(v/v) trifluoroacetic acid. The collected eluents were freeze dried and stored at -20 °C for mass spectrometry analysis.

2.2.6 Permethylation of the glycans from CD24

Permethylation was carried out according to Appendix F. in the protocol, the dried sample, typically 50 µg, was dissolved in 50 µL DMSO with a trace of DI water 15.

To this mixture 22 µL methyl iodide was added and the sample immediately placed in a spin column packed with sodium hydroxide that had been cleaned with DMSO. The sample was recovered by centrifuging at 1000 rpm for 1 min, and washed again with 100

µL DMSO. The permethylated N-glycans were recovered by extracting with 3×100 µL chloroform. The chloroform fractions were combined and washed with 6×200 µL DI water. The glycans were dried using a CentriVap® and stored at -20 °C for analysis.

2.2.7 CD24 deglycosylation with Trifluoromethane sulfonic acid (TFMS) for glycosylation site determination

For better removal of sugars, the CD24Fc was first desialylated. The sialic acids were removed by neuraminidase (Roche Applied Science, Indianapolis, IN, USA) by first having the sample dissolved in ammonium acetate 50 mM pH 5.4 followed by the addition of ~ 150 mU enzyme and incubation carried overnight at 37 °C. After this the sample was freeze dried and the deglycosylation with TFMSA carried out as described in

Appendix G. First, 1 mg of CD24 and Ribonuclease B were placed in separate glass vials and completely dried in a lyophilizer. Then, TMFS reagent was cooled to 4 °C and 150

µL added to the reaction vial and sealing it rapidly. The reaction mixture was then mixed by shaking gently for five minutes until all the glycoprotein had dissolved. Then the reaction mixture was incubated for 25 minutes with occasional shaking on ice and 4 µL of 0.2 % bromophenol blue solution added. Then dropwise, a 60 % pyridine solution that had been cooled to -15 °C in a methanol-dry ice bath was added with shaking after each drop until the reaction mixture gradually turned to yellow then blue. The reaction mixture was kept cold at -20 °C to -15 °C due to the exothermic nature of the reaction and when a

white precipitate formed, 20 µL of water was added to dissolve it. Finally the deglycosylated protein was dialyzed against 20 mM ammonium bicarbonate for purification by dialysis and the reaction monitored with SDS-PAGE and MALDI-TOF-

MS.

2.2.8 In-gel digestion of deglycosylated CD24 with protease

This was carried out as written in Appendix D. After running the gel, the band was cut from the SDS-PAGE gel and cut into small pieces of approximately 1 mm2 and washed overnight with 50 % methanol in 10 % acetic acid. The wash was repeated once more for 3h. Then the sample was washed with 25 mM NH4HCO3 in 50 % acetonitrile and dried in a SpeedVac. After this step the sample was reduced with 40 µL of 10 mM

DTT for 45 min at 60 °C, and the supernatant removed. Then 40 µL of 55 mM iodoacetamide in 25 mM NH4HCO3 was added and incubated at room temperature for 45 min in the dark and then the supernatant removed. The sample was then washed with 100

µL 25 mM NH4HCO3 for 10 min and then dehydrated by vortexing for 5 min with 2 ×

400 µL 25 mM NH4HCO3 in 50 % acetonitrile and the supernatant removed. The gel pieces were treated with 20 µL of trypsin 20 µg/mL. The sample was first allowed to incubate at room temperature for 1h and after which 20 µL of buffer was added to cover the gel pieces and the incubation carried out at 37 °C overnight. The peptides were recovered by vortexing with 40 µL 50 % acetonitrile in 2.5 % TFA, then 0.1 % TFA and finally acetonitrile each time for ten minutes. The combined extracts were concentrated in a SpeedVac and analyzed by MALDI-TOF-MS after mixing with 2-cyano-4- hydoxycinnamic acid matrix.

2.2.9 ESI-MS-MS for the glycans

A Brucker Esquire –LCMS-MS (Brucker Daltonics, MA, USA) was used for tandem mass spectrometry. For ESI, the samples were dissolved in 50 % methanol containing 1 % acetic acid and directly infused into the mass spectrometer. The nebulizing gas was set at 10 psi; the dry gas at 6 L/min; and the dry temperature at

250 °C.

2.2.10 MALDI-ToF-MS

MALDI-ToF-MS was acquired with a Kratos Axima-CFR instrument (Shimadzu,

MA, USA) and a Bruker Microflex MALDI instrument (Brucker Daltonics, MA, USA).

MALDI-TOF mass spectrometry was performed in the positive reflectron mode. The instrument was equipped with a pulsed nitrogen laser set at 337 nm. The acceleration voltage was 20 kV. The samples were normally dissolved in a 1:1 methanol/water solution containing 20 mM sodium acetate. A 10 mg/mL matrix was prepared by dissolving 2, 5-dihydroxy benzoic acid in the above solvent without the sodium acetate 15.

The sample and the matrix were mixed 1:1 before spotting on the plate and air dried.

2.2.11 LC-MS-MS for peptides

The peptides were separated on a C18 capillary column (Michrom Biosources Inc.,

Aurburn, CA) using a flow rate of 2 µL/min and detected on an LTQ-orbitrap mass spectrometer (ThermoFisher, San Jose, CA). 2 uL of sample was injected into the column and eluted with 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile(B).

The elution gradient was 2 % B for 2 minutes followed by 2-40 % B for 45 minutes. This was ramped up from 40-90 % B for 2 minutes and held at 90 % B for a further 2 minutes.

This was returned to 2.0 % B in 2 minutes and equilibrated at 2.0 % B for 15 minutes.

The heated capillary temperature and electrospray voltage was set at 175 °C and 2.2 kV respectively. All data was acquired in the positive ion mode. Full scans were acquired in the m/z range 300-2000 followed by ten data-dependent MS/MS scans with dynamic exclusion between each MS scan.

2.2.12 Data analysis

The raw MS data was converted to mzXML and searched against a human data base with an equivalent database decoy sequences using MassMatrix. The variable modifications set were the oxidation of methionine, carbamidomethylation of cysteine,

N-acetylglucosylation of serine, or/and threonine or/and asparagine and acetylation of the

N-terminal The enzyme was set depending on the one used, trypsin or chymotrypsin or

Glu-C and a maximum of two missed cleavages allowed.

2.3 Results and discussion

2.3.1 Cleavage of the CD24 from CD24IgG1 with papain and its purification

CD24IgG1 consists of the extracellular domain of the CD24 protein and the Fc fragment of IgG1. As shown in Fig. 2.1a, the purified CD24 protein is a covalent dimer.

Isoelectrofocusing revealed a complex pattern of the fusion protein, which is consistent with the extensive and diverse glycosylation of the fusion protein. In order to focus on

CD24 portion, the CD24IgG1 was cleaved with crystalline papain at the hinge region and

its fragmentation monitored with SDS-PAGE. Since no reducing reagent is present in the digestion reaction, papain yield Fc monomer and CD24 dimer16. The fragment components were purified with a protein A Sepharose column in which the Fc region of

IgG1 and the unfragmented CD24IgG1 are bound while the CD24 fragment is not. The purification was also monitored with SDS-PAGE and the gel is shown in Figure 2.1b.

The band for the CD24 glycoprotein was relatively pure at an approximate MW of 45 kDa on the non-reducing SDS-PAGE gel. This CD24 dimer was used for analysis of N- glycans.

Figure 2.1. (a). Polyacrylamide gel electrophoresis (PAGE) of the purified fused CD24 protein carried out under either non-reducing or reducing conditions. In each analysis ~2 and 5ug was loaded per well in quadruplex and the gels were stained using Colloidal Blue stain. (b). non-reducing SDS-PAGE after fragmentation with papain and purification with affinity column: line1 MW marker, line 2: the bound portion containing the Fc part of IgG in CD24IgG1, line 3: the CD24, line 4 CD24IgG1.

2.3.2 Matrix-Assisted Laser Desorption Ionization Time of Flight Mass

Spectrometry (MALDI-ToF-MS) of the released O-glycans

The released O-glycans from CD24IgG were analyzed with MALDI-ToF-MS and the scan performed in the positive mode. Figure 2.2 shows the mass spectrum of O and

N-glycans released from the CD24 glycoprotein. This means that the ammonia-based beta elimination method not only releases the O-glycans, but also N-glycans 11.

Figure 2.2. MALDI-ToF mass spectrum for CD24 O-and N-glycans released by the ammonia based beta elimination method

2.3.3 Tandem mass spectrometry of the released O-glycans

The O-oligosaccharides released by the beta-elimination chemical method were analyzed with ES-ion trap MS before permethylation. The nomenclature used for the fragmentation of the glycans was the one introduced by Domon and Costello 17. In the nomenclature two types of fragmentation occur; glycosidic cleavage between the sugars

(Y and Z) and cross ring cleavage (X). The scheme of the fragmentation is shown in

Figure 2.3.

Figure 2.3. Nomenclature for the fragmentation of carbohydrates

The fragmentation provides information on the sugar sequence of the carbohydrate. In the results, the following peaks were positively identified. The peak at m/z 675.4 (Figure 2.4a) in the negative scan mode is a glycan whose structure has

2 NeuAc1Hex1HexNAc1. The CID fragmentation MS spectra (Figure 2.4b) revealed m/z ion 290 (B1) which was due to the loss of N-acetyl neuraminic acid and at 384 (Y2),

Hex1HexNAc1. This fragment represents the core 1 of the O-glycans, Galβ-1,3GalNAc.

Therefore, this peak’s structure is proposed as Neu5Acα-2,3/6Galβ-1,3GalNAc.

The structures of the released O and N-glycans from CD24 can be deduced because the glycosyltransferases and glycosidases in the ER and the Golgi apparatus have been identified. This limits the number of possible oligosaccharide structures to a smaller

number. Table 2.1 summarizes the proposed structures of the O-glycans identified from the CD24 glycoprotein.

Table 2.1. O-glycans released from CD24 by a β-elimination with subsequent permethylation Expected ion Error Proposed Sugars from CD24 Obs. Mol. Wt. molecular wt. , ppm structurea

NeuAc1Gal1GalNAc1 879.45 879.4316 -20

NeuAc2 al1GalNAc1 1240.62 1240.6054 -11 a -Fuc, Gal, -Man, -GalNAc, -GlcNAc, -NeuAc

The glycan Neu5Acα-2,3/6Galβ-1,3GalNAc as seen in the mass spectrum in

Figure 2.4 was the most abundant oligosaccharide from CD24 with the other O-glycan from the glycoprotein being NeuAc26Galβ-1,3GalNAc. The O-glycan identified as

Neu5Acα-2, 3/6Galβ-1,3GalNAc is also called sialyl-T antigen. We report the presence of sialyl-T antigen in CD24. Mucin-like carbohydrates which include sialyl-T antigen, Tn, sialyl-Tn, and T antigen have been found to be expressed in some benign and malignant cancer cells. Moreover, sialyl-T and T antigen can be used as biomarkers in progression of several carcinomas 18-31. The antigens have been found to be expressed in gastric carcinomas such as colon cancer 19, 21, breast carcinomas 25, 32, salivary gland carcinomas

20, 27-31, cancer of the human cervix 18, pancreatic tumors 24, ovarian tumors 26 and skin tumors 22, 23. Since many of these cell types also over-express CD24 9, 33, 34, it is intriguing that the CD24 over-expression may account for the increased sT in the cancer cells.

2.3.4 N-glycans

The CD24 N-glycans that were released with the use of PNGase F were permethylated and analyzed with MALDI-ToF-MS. The mass spectrum of the oligosaccharides obtained is shown in Figure 2.5. The results showed three major peaks.

Figure 2.5. MALDI-ToF mass spectrum for the permethylated N-glycans released by PNGase F from CD24

The peak at m/z 1836.88 was identified as GalNAc2GlcNAc2Man3Fuc1, at 2041.88 was

Gal1GalNAc2GlcNAc2Man3Fuc1 and 2246.0 was Gal2GalNAc2GlcNAc2Man3Fuc1. The

N-glycans released here are similar to those released with the ammonia based beta elimination method (Figure 2.2). The N- glycans that were identified from Figures 2.2 and 2.5 are listed in Table 2.2 with their proposed structures.

Table 2.2. Permethylated N-glycans from CD24 released by PNGase F and ammonia based beta-elimination method Exp. m/z Calculated Error, Name Proposed structurea + Na m/z + Na ppm

HexNAc4Hex3Fuc1 1835.75 1835.426 -176

HexNAc Hex Fuc 2040.30 2040.0258 -134 4 4 1 HexNAc Hex Fuc 2244.37 2244.1256 -108 4 5 1 HexNAc Hex Fuc NeuAc 2605.46 2605.2993 -61 4 5 1 1

HexNAc5Hex6Fuc1 2693.45 2693.3518 -36

HexNAc5Hex6Fuc1NeuAc1 3054.78 3054.5256 -83 a -Fuc, Gal, -Man, -GalNAc, -GlcNAc, -NeuAc

From these results, it can be observed that all the N-glycans obtained are fucosylated. Studies on the binding of selectins to fucosylated carbohydrates have shown that they act as ligands for various selectins 35, 36. These data suggest that oligosaccharides on the surface of CD24 can contribute towards the metastasis of tumors in cases where the protein is expressed.

The CD24 glycoprotein has 30 amino acids 37 with a predicted MW of 2929 Da.

Since papain cut below the hinge region, another 10 amino acids are likely to be added in the CD24 glycoprotein used in this study. From the CD24 amino acid sequence, it has four potential N-glycosylation sites and a total of sixteen serine and threonine amino acids which are potential O-glycosylation sites. As such, more than 80 % of the mass in

CD24 are oligosaccharides. Since the oligosaccharide Neu5Acα-2,3/6Galβ-1,3GalNAc, contributes more than half the total amount of the glycans in the CD24 glycoprotein, the

glycans must be attached to the several serines and threonines of the glycoprotein.

Therefore studies to determine which sites are glycan modified were carried out.

2.3.5 CD24 glycosylation site determination

2.3.5.1 Deglycosylation of CD24Fc with Trifluoromethanesulfonic acid (TFMSA)

The aim of TFMS deglycosylation is to remove all the sugars attached to the peptide backbone and have access to the peptide backbone so as to identify the glycosylation sites on the proteins38. Treatment of glycoproteins with anhydrous trifluoromethanesulfonic acid (TFMS) is very effective in preserving the core protein unlike -elimination and hydrazinolysis. It has been found to efficiently deglycosylate a variety of oligosaccharide motifs including N-linked, O-linked glycans38, 39 (except the innermost Asn-linked GlcNAc or GalNAc), collagen saccharides (Hyp linkage)40, 41 and glycosaminoglycans (GAGs) linked to the proteoglycan core42.

TFMS deglycosylation has also been found to be effective on plant, bacterial, and fungal glycoproteins that are often complex and difficult to digest enzymatically.

Although TFMS hydrolysis reaction results in minimal protein degradation, the released glycans are destroyed. It has been reported that the biological, immunological, and receptor-binding activities of certain glycoproteins are retained upon deglycosylation by this method but not for all glycoproteins.

The CD24 fusion protein was deglycosylated with TFMS and after dialysis the deglycosylation monitored with SDS-PAGE. The results are shown in Figure 2.6 and it is clear that the glycans were removed. The sample was also run on MALDI-TOF-MS and the result when compared to the deglycosylated on showed a difference of 10 KDa as

shown in Figure 2.7. The sample not treated with DTT is shown in Appendix N.1 which shows the major peak being the dimer

Figure 2.6. From L-Right: CD24Fc before, CD24Fc after 1, CD24Fc after 2, MW marker

32122

200000 Intensity

63544

0 30000 m/z 110000 C:\Documents and Settings\Wang Group\Desktop\transfers from chem dept\Chem-MALDI\CD24 protein\TFMS\21810\dtt and iodoctemadie\0_C1\1\1SLin\pdata\1\1r (08:20 02/26/10) Description: Figure 2.7. MALDI-TOF-MS of TMFS treated CD24 fusion protein before reduction with DTT

The result from the deglycosylation of the CD24 fusion protein shows that the sugars from the protein were hydrolyzed. The SDS-PAGE shows the CD24Fc protein as a smear at around 65 kDa and the deglycosylated protein as clear band at about 35 kDa.

This implies that the sugar modifications on this protein account for about 10-15

KDa of the total MW. Results from MALDI-TOF-MS also revealed that the deglycosylation is successful because after the deglycosylated protein is reduced and alkylated, the MW is around 32 kDa. This molecular weight is similar to the proteins theoretical MW which is 31 kDa without any modifications. The average molecular weigh on the Fc region is 1900 Da meaning that about 8 kDa is from the O-glycosylation.

This means that the sugar on the CD24 have more molecular weight than the protein itself.

2.3.5.2 Digestion of the TFMS deglycosylated protein with proteases

2.3.5.2.1 Trypsin

The TFMS deglycosylated sample was digested with proteases (trypsin, chymotrypsin and Glu-C) to determine the glycosylation sites. Theoretically there are four potential N-glycosylation sites with three on the CD24 and the fourth on the fusion part. For O-glycans, half of the CD24 protein has serines and threonines which are potential sites. After the protease digestion, the resulting peptides were analyzed with

MALDI-TOF-MS and LTQ-orbitrap MSMS after C-18 zip tip desalting. The MALDI-

TOF-MS spectrum after digestion with trypsin is shown in Figure 2.8. The spectrum showed the presence of the peptide with N-glycosylation site, EEQYNSTYR.

Figure 2.8. MALDI-TOF-MS spectrum TFMSA deglycosylated CD24FC tryptic peptides

This peptide is modified with N-acetylglucosamine which is a monosaccharide residue that is linked to asparagine and cannot be hydrolyzed by TFMS under the reaction conditions used. The additional MW due to this monosaccharide residue is 203.1 resulting in an m/z value of 1392.3. The other peptides that were identified on this spectrum are summarized in Table 2.3. From this spectrum, only one N-glycosylation site was observed which is on the fusion region. The other N-glycosylation sites and the O- glycosylation sites were not observed probably due to the large size of the tryptic peptide.

This peptide, LGLGLLLLALLLPTQIYSSE TTTGTSSNSSQSTSNSGLAP

NPTNATTKPK was not observed. The reason could be either the peptide is too large and

is retained in the C-18 material during purification or its ionization is very weak and therefore cannot be observed in the MS spectrum.

Table 2.3. Summary of CD24 fusion protein tryptic peptides observed after analysis with MALDI-TOF-MS Theor. m/z Error, position Art. Mod. Art. Mod Peptide sequence (M+H)+ ppm mass 2744.2456 135 257-279 Cys_CA 2801.267 WQQGNVFSCSVMHEAL M: 265 2760.240 HNHY TQK MSO: 268 2730.4145 123 63-88 Cys_CA 2844.457 THTCPPCPAPELLGGPS M: 66, 69 VFL FPPKPK 2544.1313 240 211-232 GFYPSDIAVEWESNGQP ENN YK 1873.9218 68 233-249 TTPPVLDSDGSFFLYSK 1808.0064 83 142-157 VVSVLTVLHQDWLNGK

1677.8019 56 115-128 FNWYVDGVEVHNAK 1286.6739 23 185-195 EPQVYTLPPSR 1189.5120 12 133-141 GlcNAc: 1392.59 EEQYNSTYR 137 1104.6081 23 201-210 Cys_CA 1161.629 NQVSLTCLVK M: 207 838.5032 76 167-174 ALPAPIEK 835.4342 79 89-95 MSO: 92 851.4291 DTLMISR 788.4512 10 280-287 SLSLSPGK 605.3141 42 196-200 DELTK

The peptides were also analyzed on the LTQ orbitrap MS with C-18 RP separation to unambiguously identify the peptides after MSMS run. The peptides search was carried out with MassMatrix software43 (http://www.massmatrix.net) using a customized database that contains our targeted protein. The result obtained was able to identify the protein with a sequence coverage of 68 %. The coverage was only after the

CD24 peptide in the fusion region meaning that again that this peptide was too large to be

observed. Therefore to obtain a shorter peptide in the region, the protein was digested with Chymotrypsin.

2.3.5.2.2 Chymotrypsin

The chymotrypsin peptides were also desalted with a C-18 zip tip as with trypsin and analyzed with the LTQ orbitrap MS. The result obtained had a coverage of 46 % which was lower coverage than the tryptic peptides but the N-glycosylation site on the fusion part was observed. The MSMS spectrum of the peptide

VDGVEVHNAKTKPREEQYNSTY is shown in Figure 2.9. The fragmentation shows the presence of the GlcNAc modification of the asparagine by the addition of 203.1 Da to the peptide on the asparagine.

Figure 2.9. VDGVEVHNAKTKPREEQYNSTY Chymotrypsin digested CD24Fc peptide MS2 fragmentation spectrum,

Since trypsin and chymotrypsin did not provide the targeted protein, the sample was deglycosylated again with TFMS so that any remaining sugars on the protein are completely removed. After this reaction, the deglycosylated protein was digested with chymotrypsin which could provide shorter peptides in the targeted region than trypsin.

The data obtained from the LC-MS/MS data was loaded into the MassMatrix search software and yielded coverage of 46 %.

In the coverage, the peptide SSETTTGTSSNSSQSTSNSGL that is part of the

CD24 was observed. However, this peptide did not have any modifications which might imply that the deglycosylation was complete and therefore did not leave the N- acetylgalactosamine linked to the serines or threonines or N-acetylglucosamines to asparagine. The data search obtained geve the following aminoacids with the colored ones being the ones identified GRAMVARLGL GLLLLALLLP TQIYSSETTT

GTSSNSSQST SNSGLAPNPT. The MS/MS data of the peptide is shown in Figure 2.10.

Figure 2.10. MS2 data of SSETTGTSSNSSQSTSNSGL from chymotrypsin digest of CD24 fusion protein

The protein digestion with Glu-C gave coverage of 36 % and no new peptides were observed therefore was not useful in providing new information on the CD24 peptide or it glycosylation sites.

2.4 Conclusions.

The sugar structure analysis on CD24Fc was carried out with Mass spectrometry and the results revealed the presence of both N- and O-glycans. The major sugars on this protein were O-glycans due to the fact that it has many potential O-glycosylation sites.

The analysis also revealed this with the sialyl-T antigen, Neu5Acα-2,3/6Galβ-1,3GalNAc

being found to be the most abundant glycan. It appears to contribute more than half of the total glycans from CD24. The theoretical molecular weight of CD24s 30 amino acids is just 2.9 kDa. This means that this O-glycan is attached to several serines and threonines on the peptide backbone to make up to the 28-32 kDa weight that this glycoprotein is thought to be.

The determination of the glycosylation sites on the CD24Fc was carried out after sugar hydrolysis with TFMS followed by protease digestion. Analysis was carried out by mass spectrometry. The results revealed the presence of the N-glycosylation site on the

Fc region with the observing of the peptide EEQYNSTYR+ GlcNAc at m/z 1392.3. The peptide was identified due to the presence of the modification from the innermost N- acetylglucosamine of N-glycans that is attached to the asparagine on the peptide backbone.

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CHAPTER 3 Glycan structure analysis of Fea1 from Chlamydomonas reinhardtii

(Algae)

3.1 Introduction

3.1.1 Fe-assimilating protein 1 (Fea1)

Fe-assimilating protein 1 (Fea1) is a high-CO2 inducible, periplasmic protein in

Chlamydomonas reinhardtii with 361 amino acids and has a molecular weight of 37941

Da. Its code in the genbank is (NCBI) AB042098 (gene) or BAA94959 (protein). The expression of this gene has been found to be due to the deficiency of iron 1, 2 and it has been hypothesized that the Fea1 proteins are responsible for iron assimilation in

Chlamydomonas reinhardtii3.

The selection of this species of algae Chlamydomonas reinhardtii for study is due to the fact that it can be used to express therapeutic proteins. The benefits of utilizing algae for the production of therapeutic proteins are that it can be done in large amounts cheaply compared to animal sources and can be genetically or physically contained preventing escape of transgenic organisms or their introduction into human food chains,

It also is non-toxic, non-pathogenic and does not harbor animal pathogens. Also algae is a eukaryote and thus has the ability to post-translationally modify proteins since it has a common ancestry as higher plants therefore it is thought to express glycosylation which are important for therapeutic proteins which are mostly N-glycosylated.

However, the glycosylation of Chlamydomonas reinhardtii is not well known.

Only the presence on hydroxyproline rich glycoproteins (HRGP) have been identified in carbonic anhydrase4, 5. The presence of human like glycoprotein have not been located in studies carried out on algae proteins. Therefore studying the glycosylation of these target protein Fea1 will aid in setting goals for humanization of therapeutic protein to be expressed in Chlamydomonas reinhardtii.

From the amino acid sequence, Fea1 has four potential glycosylation sites at asparagines, 41, 57, 90, and 158. Therefore this study was carried out to find out if this protein is N-glycosylated and also if these potential N-glycosylation sites are modified.

3.2 Experimental

3.2.1 Materials

Fea1 was expressed in Chlamydomonas reinhardtii and kindly provided by

Professor Richard Sayre of the Ohio state University. Glycodetection kit, periodic acid, dansyl hydrazine, sodium acetate, DMSO (Sigma), rocker table (Labnet national company), Fluorescent UV-transilluminator, peptide-N-glycosidase A (Roche), C-18 cartridges (Waters corporation)

3.2.2 Glycoprotein (Fea1) detection

Gel electrophoresis using standard techniques was performed for the samples of

Fea1, BSA (negative standard), ovalbumin and transferrin (positive standards). Then the gel was removed from the cassette and transferred to a minigel wash/staining tray. The proteins were fixed in the gel by adding 100 ml of a fixing solution (3 % acetic acid with

50 % methanol) and mixing for 60 minutes at 50-60 rpm on an orbital mixer or rocker table. The fixing solution was discarded and the gel washed twice with 100 mL of water each for 30 minutes.

After this, the gel was incubated in 100 ml of the Oxidation Solution (1 % periodic acid) for 20 minutes with constant gentle mixing. The gel was protected from light during the oxidation and the steps that followed. After the oxidation, the gel was washed in 100 mL of water for 5 minutes. Then the gel was submerged in freshly prepared staining solution (0.5 mg of dansyl hydrazine dissolved in a buffer containing

50 mM sodium acetate, pH 4.75, with 500 mM NaCl, 10 % ethanol, and 5 % DMSO) covered and incubated for 60 minutes with constant gentle mixing. After this, the staining solution was discarded and the gel washed 2 × 30 minutes with 100 mL. Then the gel was removed and viewed with a standard fluorescent UV-transilluminator with emission at

312 nm. The labeling scheme for glycodetection is shown in Figure 3.1.

Periodate oxidized Labeled Glycoprotein Glycoprotein Glycoprotein O

O Periodic acid O dansyl hydrazine R R N N N(CH ) HN NH OHOH O O 3 2

SO2 O2S

SO2NHNH2

N(CH3)2 N(CH3)2

Figure 3.1. Labeling scheme for glycodetection with UV illumination

3.2.3 The release of N-glycans from Fea1 using PNGase A

In the procedure, the proteins, 0.2 mg Fea1 was treated with trypsin from

Promega using the protocol outlined in Appendix D. The reaction was quenched by boiling the reaction mixture in water for five minutes and then freeze dried. The dried sample then was dissolved in 100 µL citrate-phosphate buffer pH 5.0 and 0.5 mU of the

PNGase A enzyme added. The mixture was then incubated for 24 h at 37 °C. The samples were purified by a C-18 cartridge and the glycans permethylated following the procedure described in Appendix F.

3.2.4 Separation of PNGase A released glycans from peptides with C-18 cartridges

The cartridge was connected to a 10 mL syringe and first wetted with 1 mL HPLC grade methanol. Then it was equilibrated with 5 mL 5 % acetic acid and the sample introduced in the sample with flow through being collected. The glycans were eluted with

5 mL 5 % acetic acid, combine with the flow through and freeze dried and stored at -

20 °C for further analysis.

3.2.5 Monosaccharide analysis of Fea1 with GC-MS

The protocol for the analysis is listed in Appendix H. In the analysis, 200 µg of

Fea1 was hydrolyzed by addition of 250 µL 2M TFA and heating the mixture at 120 °C for 2h. Then the mixture was dried in gentle warming in presence of Nitrogen gas and dried further after addition of 2 drops methanol. The hydrolyzed sample was extracted by addition of 1 mL water and 1 mL hexane. The water layer was removed and dried as described above. After this the samples and standards were reduced with 100 µL solution

of NaBH4 in a 1:1 solution of ammonia and ethanol at room temperature. The reaction was stopped with the addition of 1 drop of glacial acetic acid and dried twice with the addition of two drops of 10 % acetic acid in methanol and twice with the addition of 2 drops of methanol. For peracetylation, 100 µL of acetic anhydride was added and the reaction carried out for 1h at 120 °C. After this step, the reaction was stopped with the addition of 1mL water and the alditols acetates extracted twice with 1 mL chloroform.

The extracted sample was stored for further analysis. The reaction Scheme for the peracetylation is shown in Figure 3.2.

O - O HO HBH3 HO O H OH O 2M TFA OH NaBH H H HO HO OH 4 HO OH HO O OH O OH HO O HO OH OH HO H O O O Arabinose as example e HC O C CH3 id O dr y O nh H C C O CH a 3 O ic et ac HC O C CH3 O

HC O C CH3 O

H2C O C CH3 arabinotol

Figure 3.2. Reaction scheme for sugar hydrolysis and peracetylation

3.2.6 GC-MS analysis

The alditol acetates were separated and quantified on a 5 % diphenyl, 95 % dimethyl polysiloxane 30 meter long column, 0.25 mm diameter, and 0.25 µm thickness stationary phase. The GC-MS instrument (Thermo Finnigan), was fitted with an

Electronic Impact MS. High purity helium gas was used at a flow rate of 7 mL/minute.

The separation conditions used were follows, after sample injection typically 1 µL, the temperature was maintained at 50 °C and increase to 150 °C at 30 °C/minute then ramped up to 300 °C at a rate of 5 °C/minute and maintained at this temperature for 1 minute.

The sample was dissolved in anhydrous chloroform.

3.3 Results and Discussion

3.3.1 Glycodetection of the Fea1 from Chlamydomonas reinhardtii results

Figure 3.3 shows a commassie blue stained SDS-PAGE gel of Fea1 and the major band is at ~ 43 kDa. After carrying out the glycodetection experiment, the results obtained showed that the Fea1 is glycosylated. This is observed in Figure 3.4a which shows the image of the western blot in a UV illuminator and Figure 3.4c the gel after staining.

Figure 3.3. SDS-PAGE of the unpurified Fea1. It has two bands at ~43 and 38 kDa

Figure 3.4. (a) UV photo after transfer to the PVDF membrane; b) UV photo of the SDS- PAGE gel; c) the photo of the gel. BSA, OV (ovalbuminT (transferrin) MW (molecular weight marker Fv and Fr are the Fea1 protein. R and V is part of the Fea1 protein (Fea1 band is part of the Fea1 protein (Fea1 band is circled)

In the experiment, BSA was used as a negative control while ovalbumin and transferrin were used as positive control since they are known to be glycosylated. From the images after staining it is shows that Fea1 is glycosylated since there was illumination on both Fv and Fr which are the Fea1 proteins. Therefore further analysis was carried out to identify the kind of sugars structures on Fea1 specifically N-linked sugars.

3.3.2 Fea1 monosaccharide analysis with GC-MS

The samples were per acetylated and run on a Thermo Finnigan GC-MS; the retention times obtained for the released monosaccharide alditols acetates were compared to those of monosaccharide standards. The monosaccharide standards used were mannose, glucose, galactose, xylose, fucose and N-acetylglucosamine. The GC-MS chromatogram is shown on Figure 3.5 for standard monosaccharide residues and Figure 3.6 for the Fea1 hydrolyzed sugars.

Figure 3.5. GC-MS chromatogram of alditol acetates from sugar standards mixture

Figure 3.6. GC-MS chromatogram of monosaccharide alditol acetates hydrolyzed from Fea1

These results show that there is an absence of N-acetyl glucosamine which could mean that Fea1 is not N-glycosylated the way higher plants are.

3.3.3 MALDI-TOF Mass spectrometry of the released Fea1 Glycans

The permethylated glycans were spotted on the MALDI plate and analyzed. The spectrum is shown in Figure 3.7. The results showed the presence of five possible glycan structures. The interesting thing however was that glycans number 1, 3 and 5 lacked one

GlcNAc to the pentasaccharide core. These structures were determined by using the

Consortium for Functional glycomics (CFG) database and these result shows that the sugars are glycosylated which does not happen for plants of which algae is related to.

However xylose was observed which is a sugar that is normally present in plants but not in mammals6.

Figure 3.7. MALDI-TOF-MS of the Fea1 permethylated N-glycans released by PNGase A

The released glycans identified in the mass spectrum are summarized in Table 3.1.

Table 3.1. Permethylated N-glycans from Fea1 m/z for permethylated (+ Na) monosaccharides

1 1287.35 Hex3HexAc1Sia1 2 1332.23 Hex3HexNAc3Xyl1 3 1491.49 Hex4HexNAc1 Sia1 4 1536.55 Hex4HexNAc3Pent1 5 1695.48 Hex5HexNAc1Sia1

3.3.4 Determination of the glycosylation sites of Fea1

10 µg of the sample was loaded into and SDS-PSGE gel ran and stained. After which the band was excised, it was digested with trypsin using the in-gel digestion protocol described in Appendix D. The purpose of this analysis is to identify which peptide with possible N-glycosylation sites are missing. This could mean that they are possibly glycosylated and by using tandem MS we can identify them from the fragmentation which might have sugar residues.

3.3.4.1 Analysis of tryptic peptides with MALDI-TOF-MS

The samples were desalted with a C-18 zip tip and analyzed with MALDI-TOF-

MS. The results obtained are shown in the Figure 3.8 which showing the mass spectrum of the peptides.

3000 Intens. [a.u.] Intens.

2500

860.936

2000 2068.837

1500

1435.470

1000

1755.593 1071.983 2612.038 1517.491 2291.405

500 1594.633 1355.589 1902.735

0 1000 1500 2000 2500 3000 m/z

Figure 3.8. MALDI-TOF-MS of Fea1 tryptic peptides

These m/z peaks corresponding to Fea1 tryptic peptides were identified by comparing to the expected peptides using the PeptideMass software from http://ca.expasy.org/. The PeptideMass peptides and those identified form the MS spectrum are listed in Table 3.2. The results revealed that three peptides with large molecular weight were not observed. These peptides had the larger m/z’s and of the four peptides with potential N-glycosylation sites two were observed (at m/z 2737.1982 and,

5910.7761) and two were not observed (at m/z 2290.1713 and 1902.9265). Even those observed were in very low abundance which could mean that there still is some with some glycosylation. Two large peptides without the potential of being N-glycosylated were also not observed.

Table 3.2. Identification of Fea1 tryptic peptides after MALDI-TOF mass spectrometry with PeptideMass software mass Error, position Art. Mod. Art. Mod Peptide sequence ppm mass 5910.7761 NO a 132-191 Cys_CAM 5967.7976 TYLADGTGN*LTNLVS : 170 5926.7711 DASGAPHNVDEAWAL MSO: 184 WAGGAANNCGLSGWA SSLGAAMGTTFLGK 2737.1982 NO 9-32 Cys_CAM 2794.2197 FEGFSYAGNVIGYVN* : 29 2769.1881 MTMDY CDIK MSO: 24, 26 2320.2580 NO 229-252 Cys_CAM 2377.2795 LLTLLGLQGVSVAAYT : 251 ADAA AACK

2290.1713 65-86 FASYITAN*GSVEPLHD -45 SILA GK 2069.0154 192-210 MSO: 2101.0052 SYVNTAMINTVNEMLA 86 198, 205 AAR 1902.9265 33-51 MSO: 35 1918.9215 AAMAAGN*FTEALSIYS 100 TGK 1802.9332 263-277 MSO: 1834.9230 TMIAVHWAYLEPMLK 140 264, 275 1592.8391 101 283-297 ASAVTELHHQLTASK 1517.7019 138 118-129 YHLHEVDEAYNK 1435.7539 197 211-223 LSTLNIQAYDAAR 1355.8256 174 104-117 ATLAAGLIQVGTLK 1085.5585 -312. 253-262 RPAAEVEDAK 1048.5269 9 87-96 DTSSLDAAIR 861.4425 587 1-8 QPTTTGTR *is potential N-glycosylation site, a: not observed

The list of the observed peptides is summarized in Table 3.2. To unambiguously identify these peptides, as described in Chapter 1 the peptides were analyzed with an

LTQ-MS-MS to get the fragments of the peptides. The separation was carried out with a

C-18 column with a similar trap. The data collected was searched with mass mascot and the data revealed sequence coverage of 51%. In the sequence, it was evident that large peptides were not observed as well as small ones. Also some of the peptides that had been identified in the MALDI-TOF-MS run (FASYITAN*GSVEPLHDSILA GK,

AAMAAGN*FTEALSIYSTGK,) were not observed. This means that the peaks on the

MALDI-TOF-MS could have been in very low amounts.

To obtain more coverage, the sample was digested with chymotrypsin and the digests analyzed in with the LTQ MS as outline above. The coverage obtained after

Mascot search was 44 % and the N-glycosylation site peptides were also not observed.

The Mascot search results for trypsin and chymotrypsin digests after LC-MS-MS are shown in Figure 3.9.

1 MSVGFLVLAL GALVVATAQP TTTGTRFEGF SYAGNVIGYV N*MTMDYCDIK 51 AAMAAGN*FTE ALSIYSTGKN SFSGLARRTF FRFASYITAN* GSVEPLHDSI 101 LAGKDTSSLD AAIRAALADG KATLAAGLIQ VGTLKYHLHE VDEAYNKIKT 151 YLADGTGN*LT NLVSDASGAP HNVDEAWALW AGGAANNCGT LSGWASSLGA 201 AMGTTFLGKS YVNTAMINTV NEMLAAARLS TLNIQAYDAA RTNEVRLLTL 251 LGLQGVSVAA YTADAAAACK RPAAEVEDAK TMIAVHWAYL EPMLKLRNFK 301 ASAVTELHHQ LTASKLSYKK VAAAVKGVLS AMGRRSSELG APQSAIIAAN 351 WKCSSKTLRS IA Trypsin digest gives coverage of 51 %

1 MSVGFLVLAL GALVVATA QP TTTGTRFEGF SYAGNVIGYV N*MTMDYCDIK 51 AAMAAGN*FTE ALSIYSTGKN SFSGLARRTF FRFASYITAN* GSVEPLHDSI 101 LAGKDTSSLD AAIRAALADG KATLAAGLIQ VGTLKYHLHE VDEAYNKIKT 151 YLADGTGN*LT NLVSDASGAP HNVDEAWALW AGGAANNCGT LSGWASSLGA 201 AMGTTFLGKS YVNTAMINTV NEMLAAARLS TLNIQAYDAA RTNEVRLLTL 251 LGLQGVSVAA YTADAAAACK RPAAEVEDAK TMIAVHWAYL EPMLKLRNFK 301 ASAVTELHHQ LTASKLSYKK VAAAVKGVLS AMGRRSSELG APQSAIIAAN 351 WKCSSKTLRS IA Chymotrypsin digest coverage-44 % excluding signal peptide.

Figure 3.9. Mascot search results of tryptic peptides (top) and chymotryptic peptides (bottom) of Fea1. In-gel digestions of the sample were carried out for both.

These results reveal that the absence of the N-glycosylation site peptides may mean presence of glycosylation. However, some of the peptides like

TYLADGTGNLTNLVSDASGAPHNVDEAWALWAGGAANNCGTLSGWASSLGAA

MGTTFLGK, FASYITAN*GSVEPLHDSILAGK, and, AAMAAGN*FTEALSIYSTGK for tryptic peptides could be too large and not easy to ionize. And therefore will require

other analytical methods to identify. For chymotryptic peptides, the lack of the peptide having these sites could not be explained since most of them are of reasonable size. This could therefore mean that glycosylation is a possibility. And this glycosylation could be different from the normal plant-like N-glycosylation since from the GC-MS study of the monosaccharide alditols acetates, GlcNAc residue was not observed.

3.4 Conclusions

Fea1 was analyzed for any N-glycosylation and the first step was by detecting for presence of glycosylation on the protein. The method that was used was glycodetection by chemiluminiscence. The result obtained showed that Fea1 was glycosylated since the

Fea1 band was fluorescent active after labeling with a fluorescent tag. To analyze the types of monosaccharides present on this protein monosaccharide analysis with GC-MS was carried out. The results obtained showed the presence of mannose, galactose and arabinose. However, the presence of N-acetylglucosamine was not observed. This monosaccharide residue is the one that is normally present in mammal N-oligosaccharide.

This therefore means that the oligosaccharides present on this glycoprotein might not be mammalian like N-glycans and higher plants.

Release with Peptide-N-Glycosidase A enzyme, revealed presence of some glycans which had some similarity to structure of glycans from plants due to the presence of Pentoses, hexoses and N-acetyl hexosamines. But presence of sialic acid is makes it different from the normal plant glycans. Also some of the structure did not have the normal structures of the N-glycans. The structures however were not conclusively derived.

To identify if any of the potential N-glycosylation site were modified; those with the motif N-X-T/S, the protein was digested with trypsin and chymotrypsin and the peptides analyzed with MALDI-TOF-MS. The results revealed that two peptides were missing which could mean that they are modified. The other two, were in low abundance which could not rule out incomplete modification of these asparagines. Analysis with

LTQ MS did not show presence of any of these four potential N-glycosylation sites peptides which could mean they are modified. However this modification is not plant-like due to the absence of GlcNAc residue from the monosaccharide analysis with GC-MS.

References

(1) Herbik, A.; Bolling, C.; Buckhout, T. J. Plant Physiol 2002, 130, 2039-2048.

(2) Rubinelli, P.; Siripornadulsil, S.; Gao-Rubinelli, F.; Sayre, R. T. Planta 2002, 215,

1-13.

(3) Allen, M. D.; del Campo, J. A.; Kropat, J.; Merchant, S. S. Eukaryot Cell 2007, 6,

1841-1852.

(4) Shaw, P. J.; Hills, G. J. J Cell Sci 1984, 68, 271-284.

(5) Bollig, K.; Lamshoft, M.; Schweimer, K.; Marner, F. J.; Budzikiewicz, H.;

Waffenschmidt, S. Carbohydr Res 2007, 342, 2557-2566.

(6) Lerouge, P.; Cabanes-Macheteau, M.; Rayon, C.; Fischette-Laine, A. C.; Gomord,

V.; Faye, L. Plant Mol Biol 1998, 38, 31-48.

CHAPTER 4 : Glycosylation analysis of recombinant Tumor Necrosis Factor

Receptor.

4.1 Introduction

4.1.1 Tumor necrosis factor (TNF)

Tumor necrosis factor (TNF) is a multifunctional proinflammatory cytokine that is produced by different kinds of cells which plays a key role in the defense of the host and immunosurveillance1-3. It normally can cause cell death by acting through the tumor necrosis factor receptor. There are two types of TNF, TNF- and TNF- with the former being the most well-known. The TNF- apart from the role described above, it can also inhibit viral replication and tumorigenesis. However its deregulation can cause cancer4

TNF- is inhibited by interleukin 10 and is also known as lymphotoxin.

4.1.2 Tumor necrosis factor receptor (TNFR)

Tumor necrosis factor receptor (TNFR) is a cytokine receptor that binds tumor necrosis factor (TNF). This glycoprotein has been approved as a therapeutic protein in the

Unites States and it is commercially called Enebrel or Etanercept. In therapy, it is used for treatment of rheumatoid arthritis in patients not responding to other therapeutic strategies. In addition, it is used to treat moderate to severe juvenile

rheumatoid arthritis and psoriatic arthritis. The mechanism of action for Enebrel is by sequestering of TNFα.

This protein was expressed in in Camelina sativa which is a non-food, oil seed crop that is highly self- pollinating that produces seeds with a high protein (25%) and oil

(40%) content. It is an excellent seed producer with nopotential for entering the food supply or cross-pollination. In addition the seeds can be stored for future use and therefore a very attactive mode for expressing therapeutic protein

4.2 Experimental

4.2.1 PNGase F and PNGase A digestions

This glycoprotein was expressed in Camelina sativa was kindly donated by

Professor Eric Murphy from the University of North Dakota. This protein consists of the tumor necrosis factor receptor fused with the Fc region of human IgG. The glycoprotein was first digested with PNGase F to release the mammalian like N-glycans that is those that don’t have the α-1,3 Fucose linked to the first GlcNAc after asparagine. The method followed here is described in Appendix D. In another experiment, the glycoprotein was analyzed by PNGase A also described in Appendix D and the released glycans analyzed by MALDI-TOF-MS after permethylation as described in Appendix F.

4.3 Results and Discussion

The MS spectra for the permethylated sample after digestion with PNGase A is shown in Figure 4.1. From the spectrum, seven N-glycans were observed at m/z 1506,

1577.31, 1662.3, 1822.48, 1988.60, 1995.10 and 2192.77. These peaks were identified

using the CFG database based on the molecular weights observed from MALDI-TOF-MS.

This result showed two peaks with fucosylation and three with xylosylation. Two of these peaks (at mz 1506 and 1995) were fucosylated and xylosylated while one had only

Figure 4.1. MALDI MS spectrum of the permethylated N-glycans released from TNFR/Fc using PNGase A

xylosylation (m/z 1822.48). No peak had fucosylation only. The result for the sample digested with PNGase F and permethylated, is shown in Figure 4.2. The spectrum revealed five N-glycans and all the peaks identified were also observed in Figure 4.1. The only two seen in Figure 4.1 but not in 4.2 were the ones that had fucose implying resistance to digestion with PNGase F.

Figure 4.2. .MALDI MS of permethylated glycan form TNFR/FC using PNGase F only

These data revealed that the fucosylation of the N-glycans from this glycoprotein are plant-like not mammalian since no structure with fucose was revealed when released with PNGase F. Therefore the N-linked glycan structures of TNFRFc were plant-like due to the presence of α,1-3 Fucose and α1-2 xylose attached to the first mannose. The presence of these sugars normally will cause immunogenic reactions in humans since humans have antibodies against these sugar epitopes. This study therefore showed that an expression process needs to be worked out to eliminate these immunogenic sugars for the plant Camelina sativa to be used in expression of TNFRFc.

4.4 Conclusions

TNFR is a protein that is currently used in arthritis treatment in the USA. The market name is Eternercept. This chapter details the experiment in analyzing the glycosylation on this protein that was expressed in Camelina sativa. Expression of

therapeutic proteins will be advantageous since they have many advantages as outlined in the introduction of this chapter.

Our analysis revealed that the N-glycosylation modification on this protein was plant-like. This is because of the presence of α,1-3 Fucose which was present only when

PNGase A was used but absent when PNGase F was used. Also present was the pentose xylose which is normally present in plant N-glycan. Identification of these sugars is important because their presence on TNFR-Fc will cause immunogenic reaction when injected into humans.

References

(1) Shirai, T.; Yamaguchi, H.; Ito, H.; Todd, C. W.; Wallace, R. B. Nature 1985, 313,

803-806.

(2) Pennica, D.; Nedwin, G. E.; Hayflick, J. S.; Seeburg, P. H.; Derynck, R.;

Palladino, M. A.; Kohr, W. J.; Aggarwal, B. B.; Goeddel, D. V. Nature 1984, 312, 724-

729.

(3) Aderka, D. Cytokine Growth Factor Rev 1996, 7, 231-240.

(4) Locksley, R. M.; Killeen, N.; Lenardo, M. J. Cell 2001, 104, 487-501.

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CHAPTER 5 : Paraoxonase (PON1, the G3C9 Variant) Expression and PEGylation

for Bioscavenger Studies.

5.1 Introduction

5.1.1 Paraoxonase

5.1.1.1 History

Paraoxonases are types of enzymes that hydrolyze organophosphates and esters.

The first report of an enzyme in animal tissue capable of hydrolyzing organophosphate compounds was in 19461 and the enzyme paraoxonase 1 in humans was first identified in the early 1950s2, 3. These enzymes are called paraoxonase due to their ability to hydrolyze an insecticide called paraoxon whose structure is shown in Figure 5.1.

O P O NO2 H3C O

CH3

Figure 5.1. The structure of paraoxon

5.1.1.2 Types of paraoxonases

There are 3 known members of paraoxonases, PON1, PON2 and PON3 and they are located on the long arm of chromosome 7q21-224. These enzymes share structural properties and enzymatic activities. The genes PON1 and PON3 are synthesized in the

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liver and the kidney and their proteins are found in circulation bound to the high-density lipoprotein (HDL). PON1 retains its hydrophobic lead sequence which is required for

PON1’s association with HDL5-7. For PON2, its genes are expressed in the tissues including brain, testis, liver, and kidney. PON2’s protein products are not found in the plasma since this is an intracellular protein8.

5.1.1.3 Human PON 1 features and structure

Human PON is a 355-aminoacid, calcium-dependent enzyme, with a molecular weight of 43-45 kDa and has two N-glycans9. Four polymorphisms in the promoter and coding regions of the human PON1 gene (–107CNT, –162ANG, –824GNA, –907GNC) have been reported to have an effect in the gene expression and thus the concentration of

PON1 in blood10. They also have an effect on the enzyme activity. The 3D dimensional structure has not yet been dissolve. However the X-ray structure of a hybrid mammalian recombinant PON1 was recently determined11 which is very similar to human PON1. The model of the structure is shown in Figure 5.2.

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Figure 5.2. Model of the structure of huPON1 based on the X-ray crystal structure of a chimeric rabbit PON1 variant. Important residues are rendered as sticks and colored for: Ca2+ binding (red and purple); putative sites of glycosylation (green); cysteines (orange); and putative site of HDL binding (yellow). Blue and purple residues have been found to affect the activity or specificity of PON1 in derivatization, mutational or evolutionary studies. The preponderance of the activity and structural evidence suggests that the active site lies in a pocket just above (and probably including) the Ca2+ binding site in the middle of the β-barrel. The model was generated with SwissModel from PDB entry 1V04, and the figure was generated with PyMOL.

From the structure PON1 has two calcium ions in the center of the tunnel that is necessary for the activity of the enzyme. The Calcium ions are 7.2 Å apart with one calcium playing a catalytic role and the other structural11.

5.1.1.4 PONs functions

The physiological functions of these enzymes are not clearly known but as stated above paraoxonase 1 (PON1) is a serum hydrolase which is active against carboxyl esters such as phenyl acetate and organophosphate (OP) compounds such the nerve gasses VX and sarin and paraoxon, a pesticide12. Human PON1s (huPON1) native substrates are not known, although it has been shown to act as a lactonase in nature where it hydrolyzed

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naturally occurring lactone metabolites 13. It also has been shown to protect low-density lipoprotein (LDL) from oxidation14, 15.

5.1.1.5 Paraoxonase in health and diseases

This enzyme has been found to have an effect in several areas of health and disease. An example is the lowering of risk of developing atherosclerosis and coronary artery disease for PON1 and PON3. This is because of the association of coronary artery diseases with PON1 gene polymorphism.16, 17 PON1 activity has been found to affect aging with some individuals have been found to live longer due to its activity in which it counteracts harmful pro oxidants and pro inflammatory molecules18.

Other than the already outline disease, there are several health problems that paraoxonase has been shown to play a role in. these diseases include; lung cancer19, breast cancer20, ovarian malignancy20, gastro intestinal cancer21, and prostate cancer22, obesity a factor for diabetes and cardiovascular disease23, type II diabetes24-26, metabolic syndrome27, autism28, Alzheimer’s disease29, Parkinson’s disease30, 31, depression32, schizophrenia33, ischemic stroke34 and other diseases. In some of these diseases, PON 1 can be used as a diagnostic or prognostic marker. And also because this enzyme plays a role in these diseases it has become a target for researchers in seeking treatments for the ailments.

5.1.1.6 PON as an OP inactivator

Using of enzymes to inactivate OP nerve agents has been suggested as a strategy for treatment, with the investigation of number of bacterial and human enzymes, such as

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huPON135, bacterial organophosphorous hydrolase36, and butyrylcholine esterase

(BuChE) being done37, 38.

These enzymes are divided into two categories: catalytic bioscavengers such as

OP hydrolase, mutant BuChE, and the paraoxonases which are not inactivated after hydrolyzing OPs and stoichiometric bioscavengers which inactivated by reactions with

OPs. HuPON1 appears to be a promising therapeutic agent since it is from humans and will not elicit immune response and it is catalytic which will reduce the dosage of the enzyme. Human Paraoxonase 1 is a 40 KDa glycoprotein with two N-glycosylation sites at residues 252 and 323 with 354 amino acids. In this study, G3C9, a recombinant PON1 variant was expressed in E. coli and PEGylated for testing against OPs.

5.1.2 Organophosphate Compounds

Organophosphorus anticholinesterases (OPs) are among the most toxic substances that have been identified 39. Although OPs were originally developed for use as insecticides, 40 they were later adopted as weapons of warfare due to their extreme toxicity toward higher vertebrates41. Table 5.1 shows the structures of the OPs that have been most commonly utilized as chemical weapons (referred to as nerve agents). These are the Russian V-agent, VR, VX, tabun (GA), sarin (GB), soman (GD) and cyclohexylmethyl phosphonofluoridate (cyclosarin, (GF)). These compounds are still a threat to civilian populations as shown in the attack by the Aum Shinrikyo terrorist group in a subway in Japan in which they used both VX and sarin.

These compounds have a molecular weights range from 140 to 267 Daltons (Da), and under standard conditions they are all liquids that differ in their degrees of volatility

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42 . (They have median lethal dose (LD50) values in mammals, including estimates for humans, in the μg/kg dose range for all routes of exposure except dermal, where LD50 doses are in the mg/kg range 41.

Pretreatment with pyridostigmine bromide (PB) can be administered to those individuals that are thought to be at a high risk of exposure to the nerve agent GD. This drug will mask the active site temporarily (about 30 % of peripheral AChE molecules) and this will protect the enzyme from the inhibition of the OP agent irreversibly43. But this pretreatment is not feasible for civilians due to the unpredictability of a terrorist attack and can only be given to first responders.

In case of OP intoxication in a civilian setting, the method used for treatment is the administration of drugs to individuals that have been exposed as soon as possible to take care of the signs of the intoxication. Drugs that are administered include cholinoltyic drugs for example atropine which will antagonize the level of acetylcholine due to AChE inhibition44.

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Table 5.1. Names and structures of Organophosphate compounds used as nerve agents Name Structure

VX O N O P S VR O N O P S

GA (tabun) O O P N CN GA (sarin) O P O F GD (soman O P O F GF (cyclosarin) O P O F Thiosarin O P O F

Another drug that is given on the onset of exposure to OPs is an oxime which will restore the normal activity of the inhibited enzyme by reacting with the phosphoester of the inhibited enzyme to displace the phosphoryl group45.

Diazepam is an anticonvulsant drug that is given in case the symptoms from OP intoxication are severe. This drug will control tremors and convulsions induced by the OP.

Although these treatment regimens have shown some effectiveness in preventing lethality, they have some disadvantages like not totally preventing loss of consciousness, E behavioral incapacitation or permanent brain damage 46. For the maximum effect, these drugs also have to be administered within 10-40 minutes.

Nerve agents like sarin, cyclosarin and soman will cause complications since after inhibiting AChE, they undergo another reaction called aging in which dealkylation of the

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phosphoryl group attached to the inhibited enzyme occurs. And when this aging happens, the enzymes cannot be reactivated by oximes47, 48. From the OP mechanism, an approach to reduce the concentration of the OP before it reaches the site of action (synaptic endplates) should be effective. Therefore, the use of endogenous catalytic scavenger(s) such PON1 as detoxifying drugs is desirable since it will not require repeated drug administrations as in the case of oxime and atropine treatment and also can provide protection against post-exposure of these nerve agents.

5.1.3 Endotoxin removal from G3C9 and PEGylated G3C9

5.1.3.1 Endotoxins

Endotoxins, are a major component of the outer membrane of Gram-negative bacteria (Figure 5.3) and are normally released in large amount when bacteria is lysed or upon cell death. The other name for endotoxins is lipopolysaccharides (LPS). The location of LPS molecules is exclusively in the outer layer of the outer cell wall of Gram- negative bacteria. Here, the LPS role is to protect the cell against leakage of cytoplasmic material and harmful external components, such as detergents, antibiotics, and bile salts.

They composition of LPS is the hydrophobic lipid moiety (Lipid A) which is linked covalently to the hydrophilic polysaccharide moiety. As shown in Figure 5.4, LPS has three regions, the Lipid A, the core polysaccharides and the O-antigen. It is the lipid

A which has a conserved structure that is responsible for the toxicity and the polysaccharide is associated with immunogenicity. Lipid A is composed of β-1-6 linked

D- N-acetyl glucosamine which is linked to three 12-16 carbon atoms hydroxyl-acyl substituents via ester and amide bonds.

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Figure 5.3. Molecular model of the inner and outer membranes of E. coli K-12225 Abbreviation: PPEtn (ethanolamine pyrophosphate); LPS (lipopolysaccharide); Kdo (2- keto-3-deoxyoctonic acid)

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Figure 5.4. Chemical structure of endotoxin from E. coli O86:B4226. (Hep) L-glycerol- D-manno-heptose; (Gal) galactose; (Glc) glucose; (KDO) 2-keto-3-deoxyoctonic acid; (NGa) N-acetyl-galactosamine; (NG) N-acetyl-glucosamine

In this work the removal of the endotoxins was carried out with agarose bound polymixin B which binds the LPS.

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5.1.3.2 Endotoxin removal.

5.1.3.2.0 Methods of removal.

Several methods have been employed to remove endotoxins from biological preparations. The use of each method depends on the properties of the protein being cleaned

5.1.3.2.1 Ion -exchange chromatography.

Anionic exchange removal takes advantage of the negatively charged endotoxins which will bind to the anion exchange resin. This method used to remove toxins from positively charged proteins for example urokinase49. For negatively charged proteins, its use results in significant loss of the protein and for net-positively charged proteins, they can also drag the endotoxins along with them thus reducing the removal efficiency50.

5.1.3.2.2 Ultrafiltration;

This method is useful for proteins small proteins such as myoglobulin and in cases where the endotoxins aggregate to big molecules. However this method will not work if some of the endotoxins don’t aggregate and pass together with the product.

5.1.3.2.3 Affinity adsorbents

These include poly-L-lysine, immobilized L-histidine, poly(-methyl L- glutamate), and polymyxin B50-52. These compounds are generally immobilized on sepharose 4B or agarose and they have capabilities of binding the endotoxins. For example polymyxin B binds endotoxins via electrostatic and hydrophobic interactions.

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5.1.3.2.4 Two-phase micellar system

In this method the endotoxin is removed above the critical micelle concentration

(CMC) by which the endotoxins hydrophobic part interacts with the hydrophobic regions of the surfactants separating with the region water-phase which is the micelle-poor phase.

An example of surfactants used in this method is Triton X-11453, 54. This surfactant leads to the formation of two phases above a certain temperature one micelle-rich and the other micelle-poor55, 56. The method led to reduction of the endotoxins by 100 times to 30

EU/mg57. Other methods that have been used in endotoxin removal include gel filtration chromatography and sucrose gradient centrifugation

5.1.3.2.5 Endotoxin removal with Polymyxin B

Polymyxin B (PMB) is derived from bacteria, Bacillus polymyxus. The structure of PMB is shown in Figure 5.5 and it consists of a heptapeptide ring attached to C8-C9 fatty acid via an amide bond. The ring is formed by an amide bond between the carboxylic group of the C-terminal and the -amino group of diaminobutyric acid (DAB).

It has been found that PMB has a very high binding affinity for the lipid A moiety of most endotoxins58 by binding 1:1 to the lipid A portion of LPS. PMB is used as an antibiotic where it acts by formation of stable complex with the LPS of bacteria via hydrophobic and electrostatic interactions. This binding leads to a spontaneous incorporation of PMB to the bacteria’s membrane which increases the membrane permeability of the bacteria thus inhibiting its respiration. The electrostatic and hydrophobic interactions are from the positively-charged primary amino groups and the lipid part of PMB58.

112

 -NH2 CH3 L-DAB -D -Phe -L - Leu x H2SO4 RCH2CH(CH2)4CO-L-DAB-Thr-l-DAB-L-DAB L -Thr - L -DAB -L -DAB  -NH  -NH 2 2   -NH2 -NH2

Figure Error! 5.5. No Polymyxin text of specified B structure style in document. Polymyxin-1. Polymyxin B1 (R=CH3), B structure Polymyxin Polymyxin B2 B1 (R=CH3), (R=H), DAB=,α’,γPolymyxin B2- (R=H),diaminobutyric DAB=,α’,γ-diaminobutyric acid) acid)

5.1.3.3 Endotoxin assaying

Presently only two methods are used in assaying medicinal products for endotoxins. The LAL (Limulus amoebocyte lysate) assay which is the most commonly used59, 60 and is the one approved by Food and Drug Adminstaration (FDA), and, the rabbit pyrogen test. The use of the rabbit pyrogen test which was introduced in the 1920s is less common currently than the LAL assay due to it being costly, not quantitative and that it uses experimental animals. The rabbit pyrogen test involves the measurement of temperature rise in rabbit after intravenous injection. The principle here being that the endotoxins will produce endogenous pryogens in rabbit causing a fever.61

The principle of the LAL assay is that the endotoxin will cause coagulation of horseshoe crab blood (haemolymph)62. There are three LAL assay methods also called bacterial endotoxin tests (BET) used for endotoxin determination.

5.1.3.3.1 The LAL gel-clot assay

This is the simplest LAL assay method and low endotoxin level is achieved when no clotting is observable60, 63 since mixing the LAL with the sample containing endotoxin will clot. The clot is proportional to the endotoxin level in the sample.

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5.1.3.3.2 Turbidimetric LAL assay

It is also kinetic based and can give accurate measurement of the endotoxin. It contains enough coagulogen to be turbid but not clot64. The optical densities are measured and a standard curve drawn from which the unknown sample endotoxin concentration is determined65. Its quantitative range is 0.01-100.0 EU/mL

5.1.3.3.3 Chromogenic LAL assay

In this kinetic based method the coagulogen is replaced completely by a chromogenic substrate66. An enzyme is used to hydrolyze the chromogenic substrate will release p-nitrophenol, a yellow substance. The time taken for the p-nitrophenol to be released is proportional to the endotoxin concentration67.

For LAL assay methods their disadvantage is that they can give false positives or exaggerate the endotoxin quantities in a sample and is only applicable to bacterial endotoxins68.

5.2 Experimental

5.2.1 Materials

G3C9 strain was obtained from Professor Magliery lab of The Ohio State

University, Columbus, OH. NHS activated methoxy polyethylene-glycol (NOF corporation, Tokyo, Japan), HiTrap Chelating HP 5 mL column, Hitrap Q FF, Hiload

16/60 Superdex 200 and an AKTA FPLC System (GE Healthcare), FlexStation 3

Microplate Reader (Molecular Devices, Sunnyvale, CA, USA), Foto/Analyst FX convertible transilluminator (Fotodyne incorporated, Hartland, WI, USA), 10 % Tris-HCl

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ready SDS PAGE Gels ( Bio-Rad Hercules, CA USA). Corning Scholar 425 pH meter.

All the other chemicals were obtained from Sigma.

5.2.2 G3C9 Transformation and expression

The G3C9 strain was obtained from Professor Magliery lab in OSU. In the expression protocol described in Appendix I, PON1 was transformed in pET32-trx into

Origami B DE3 (Novagen) and grown on LB-Ampicillin -Kanamycin - CaCl2 (1 mM) plates. For a liter, the starter (seed) was grown from a single colony overnight at 37 °C in

5 mL 2xYT supplemented with 100 ug/mL Ampicillin, 15 ug/mL Kanamycin, and 1 mM

CaCl2. This starter was innoculted into 1 liter 2xYT containing 100 ug/mL Ampicillin,

15 ug/mL Kanamycin, and 1 mM CaCl2. Then it was incubated at 37 °C till an OD 600 of

~0.6, and induced with IPTG 0.1 mM at 30 °C for 3hrs. After this the cells were harvested at 6000 g for 10 min at 4 °C using a Beckman J2-21 centrifuge (Beckman

Instruments, Palo Alto, CA, USA).

5.2.3 G3C9 Purification

The harvested cells were suspended in 60 mL freshly prepared Lysis Buffer (for 1

L growth). The lysis buffer was 50 mM Tris pH 8.0, CaCl2 1mM, NaCl 50 mM, DTT 0.1 mM. Then the cell suspension transferred through the syringe with a 22G needle to aid in cell breaking and the bacteria sonicated on ice with a Vibra-Cell high intensity ultrasonic processor (Sonics & Materials, Inc, Danbury, CN, USA). The sonication program settings were: 3 min program, 30 sec at 5.5 setting, 2 min on ice. After this, 0.1 %

Tergitol was added and mixture shaken at room temperature on a tube rotator (Eberbach

Scientific Instruments and Apparatus, MI, USA) for about 2.5 hours and centrifuged at

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10,000 RPM with a Beckman J2-21 centrifuge type JA 20 rotor for 1 h at 4 °C and supernatant transferred to a clean tube.

5.2.4 G3C9 purification using Ni-NTA Column (Trx-His fusions)

The protein was purified with a 5 mL HiTrap Chelating HP column as described in Appendix J. The column was first activated with Nickel as follows; The column was first washed with 15 mL DI water to remove the storage solvent followed by loading 2.5 mL 0.1 M NiSO4. Then after the salt solution had ran out, the column was washed with

15 mL DI water. This was followed by 5 CV Activity Buffer (50 mM Tris pH 8.0, 1 mM

CaCl2, 50 mM NaCl, 0.1 % Tergitol). Then the supernatant obtained in section 5.2.3 was loaded into the column using the FPLC system. The loaded protein mixture sample was washed with 50 mL Activity Buffer followed by 50 mL Activity Buffer containing 20 mM Imidazole. The final washing was accomplished with 20 mL Activity Buffer containing 50 mM imidazole and the sample eluted with 30 mL Activity Buffer containing 250 mM Imidazole.

5.2.5 PEGylation

G3C9 was PEGylated with N-hydroxy succinamide ester activated mPEG of ~ 28 kDa. The G3C9 which was in Tris HCl was buffer exchanged to phosphate buffer pH 4.7, pH 6.0 and pH 8.8 to determine the best conditions for the PEGylation. After identifying the optimum pH, a 5mg/mL G3C9 sample was reacted with the NHS PEG (5 equivalents,

10 eq, 20 eq, 30 eq and 40 eq) and the reaction carried out at 4 °C for 48h with gentle

116

stirring to determine the optimum amount of equivalents of PEG to be used. After the reaction the samples extent of PEGylation was analyzed with SDS-PAGE.

O O O O pH ~ 8.0 Me O O N + H2N PEG N n H O Figure 5.6. NHS activated mPEG reaction with G3C9

5.2.6 Chromatographic purification of the PEGylated G3C9

5.2.6.1 Size Exclusion Chromatography

The PEG-G3C9 was buffer exchanged to 20 mM Tris buffer pH 8.0 containing 50 mM NaCl, 1 mM CaCl2 and 0.1 % Tergitol and loaded into a Hiload 16/60 Superdex 200 prep Grade column (GE Healthcare). The sample was eluted with 20 mM Tris buffer pH

8.0 containing 50 mM NaCl, 1 mM CaCl2 and 0.1 % Tergitol. 4 mL fractions were collected and the fractions containing the peaks were pooled and analyzed with SDS-

PAGE for purity.

5.2.6.2 Anion Exchange Chromatography

Purification was carried out as described in Appendix K. The PEGylated G3C9 was buffer exchanged to 20 mM Tris buffer pH 8.0 containing 50 mM NaCl, 1 mM

CaCl2 and 0.1 % Tergitol and loaded into a Hitrap Q FF anion exchange column (GE

Healthcare) on an AKTA FPLC System (GE Healthcare). Before sample loading, the column was equilibrated with solvent A (20 mM Tris buffer pH 8.0 containing, 1 mM

CaCl2). Using the FPLC, a linear gradient was used to elute the samples. The gradient

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was increased from 0-100 % B (solvent A with 1 M NaCl). 4 ml fractions were collected and the fractions with highest activity against Paraoxon analyzed for purity with SDS-

PAGE.

5.2.7 G3C9 Enzyme kinetic assays before and after PEGylation

G3C9’s activity was measured by following the rate at which it oxidized

Paraoxon (Appendix L). In the procedure a fresh solution of 0.13 M Paraoxon (in 100 %

Methanol) working stock was prepared on the day of experiment. The reactants were loaded on the 96 wells plate in the following order; Reaction Buffer (20 mM Tris buffer pH 8.0 containing 50 mM NaCl, 1 mM CaCl2 and 0.1 % Tergitol), Paraoxon and finally

10 µL of enzyme according to Table 5.2.

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Table 5.2. Reaction conditions for kinetic measurements for G3C9 using paraoxon Vol. of Vol. from 0.13 M Reaction Working Effective Total [Paraoxon] Buffer (uL) Paraoxon Stock (uL) in Reactions (mM) 189.6 0.4 0.26 188.8 1.2 0.78 188.0 2.0 1.30 187.0 3.0 1.90 186.4 3.6 2.36 186.0 4.0 2.60

Immediately after the addition of the enzyme, the rate of appearance of p- nitrophenol was monitored at OD 412 nm using a FlexStation 3 Microplate Reader.

These reactions were carried out in triplicates. The enzyme was diluted appropriately to

ODs that could be followed for 10 minutes at 20 second intervals.

After the OD measurements, the measured slope (change in absorbance per minute due to product formation) of the reaction was obtained using excel spreadsheet.

After this, the concentration of enzyme in reaction wells determined, and then using

Beer’s Law (equation 1) the change in concentration of p-nitrophenol formation per minute was determined.

Beer’s law: (1)

Where A is the absorbance; l is the pathlength; C is concentration; and ε is the

extinction Coefficient of p-nitrophenol. (The values for l = 0.596 cm and ε

-1 -1 =17000 cm M were used).

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5.2.7.1 Determination of the Kinetic constants with the Michaelis-Menten equation

For the Michaelis-Menten equation (equation 2), the Change in concentration of p-nitro phenol formed per minute (Y-axis, Initial Velocity) vs. Concentration of input substrate (X-axis, Concentration) was plotted to determine Km (concentration at ½ Vmax and Vmax (maximal velocity)). kcat was determined by dividing Vmax by enzyme concentration. Also determined was catalytic efficiency (kcat/Km)

Michelis-menten equation: (2)

5.2.8 Endotoxin removal and measurements

5.2.8.1 Endotoxin removal

The endotoxins were removed by using agarose immobilized Polymyxin B according to the manufacturer’s protocol described in Appendix M (Thermo Fisher scientific). In the protocol, the Detoxi-Gel Resin was regenerated by washing with five resin-bed volumes of 1 % sodium deoxycholate, followed by equilibration with 3-5 resin- bed volumes of pyrogen-free buffer (50 mM Tris buffer pH 7.5, 150 mM NaCl and 1 mM

CaCl2) to remove the detergent. The samples were first buffer exchanged to 50 mM Tris buffer pH 7.5, 150 mM NaCl and 1 mM CaCl2 and applied to the column. Then aliquots of pyrogen-free buffer were added and the flow-through collected. The column flow was stopped after sample has entered the resin bed, and incubated in the column for one hour before collecting the sample.

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5.2.8.2 Endotoxin assays

The endotoxin level measurements were carried out using the Genscript LAL assay endotoxin assay kit. In the detection protocol, 100 μL of standards, samples and endotoxin free water was carefully dispensed into different endotoxin-free vials with one blank. Then to this, 100 μL of LAL was added to each vial and then mixed thoroughly.

After this, the vials were placed in an incubation rack and incubated for 45 minutes in a

37 °C Oven. After the incubation, 100 μL of chromogenic substrate solution was added to each vial, mixed well and incubated for 6 minutes in a 37 °C oven. Then, 500 μL of stop solution was added to each vial, mixed followed by the addition of 500 μL of the second color-stabilizer. The final step was the addition of the color-stabilizer #3 followed by taking absorbance readings for each sample at 545 nm with distilled water as a blank using the FlexStation 3 Microplate Reader.

5.3 Results

5.3.1 G3C9 expression and purification

The sample was collected, analyzed with SDS-PAGE and the concentration measured with Bradford assay. For the 1 liter, the concentration was on average found to be ~ 20 mg. The SDS PAGE gel image after purification through the nickel column is shown in Figure 5.7. The purified G3C9 was also found to be active against paraoxon compound after assaying.

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Figure 5.7. FPLC chromatogram of the protein supernatant purification with Hitrap Chelating Nickel column and the SDS-PAGE of the collected sample fractions

From the SDS-PAGE gel, the bands of the collected sample appeared to be pure and the sample was used for PEGylation.

5.3.2 G3C9 PEGylation

The G3C9 was first PEGylated at different pH values to find the optimum pH value for the reaction. The reactions were monitored with SDS-PAGE and the results showed that the best PEGylation pH was 8.8 as shown in Figure 5.8 left. This pH was used for the rest of the reactions. First, it was used to monitor the right amount of PEG to use in completing the PEGylation. It was found that reacting with 40 equivalents of PEG appeared to the most complete (Figure 5.8 right) but due to the large size of PEG the reaction solution was very viscous distorting the SDS-PAGE runs. Therefore 10 equivalents of PEG were used for the rest of the reactions which also gave mainly 2

PEGs on each protein. The reaction were found to be complete in 30 minutes at room temperature and stored at 4 °C for purification.

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Figure 5.8. G3C9 reaction with PEG. left, pH optimization and on the right PEG equivalents optimization. S is pure G3C9 and M is molecular weight marker

The results shown in Figure 5.8 also revealed that when <20 equivalent PEG was loaded, the major product was protein with 2 PEG (Mw ~ 10 kDa) and when > 30 eq

PEG was loaded, majority rePON1 was PEGylated in 48 h with product containing more than 4 PEG on each protein.

5.3.3 PEGylated G3C9 purification

5.3.3.1 Size exclusion chromatography (SEC)

The results of the SEC chromatography is shown in Figure 5.9 and the collected peak fractions SDS PAGE in Figure 5.10 From the SDS-PAGE, it is clear that the separation did not provide very pure PEG-G3C9 since lane 4 shows the PEGylated sample which however still had the unPEGylated sample. Therefore SEC was found not able to provide a method for the PEGylated sample purification and the purification was carried out with anion exchange chromatography.

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Figure 5.9. SEC of PEGylated G3C9. The sample was loaded on HiLoad 16/60 Superdex 200 column and eluted with 20 mM Tris buffer pH 8.0 containing 50 mM NaCl, 1 mM CaCl2 and 0.1 % Tergitol at a Flow rate was 0.5 mL/min

Figure 5.10. SDS-PAGE of the SEC peaks 1. MW marker, 2. G3C9, 3. Peak 1, 4. Peak 2, 5. Peak 3

5.3.3.2 Anion Exchange chromatography (AEX)

Because no pure sample of the PEGylated G3C9 was obtained after SEC, the sample mixture was purified with anion exchange chromatography. The obtained chromatogram after the AEX and the SDS-PAGE gel for each peak is shown in Figure

124

5.11. From the result, it is clear that the PEG-G3C9 was eluted earlier than the G3C9 because the PEGylation removes some of the protein’s negative charges and also shields some on the G3C9. This is because in AEX, the more the negatively charged a sample is, the more it is retained and therefore will be eluted later. It can also be observed from the

Figure that the extent of PEGylation is not that high although for the PEGylated G3C9, commassie blue in not a good quantitative indicator. The SDS PAGE shows that The

PEG-G3C9 was relatively pure for further analysis.

Figure 5.11. Anion exchange chromatography of PEGylated G3C9, separation was carried out as described in the text. The SDS PAGE of each peak is also as shown

125

Therefore the sample collected from the peak showing pure PEGylated GC39 on

SDS-PAGE was used for kinetics analysis and endotoxin removal for pharmacokinetic studies.

5.3.4 G3C9 and PEG-G3C9 kinetic studies

The samples were reacted with paraoxon and the reaction followed by monitoring the formation of p-nitrophenol at 412 nm from paraoxons hydrolysis with G3C9. The concentrations of paraoxon used for each reaction are shown in Table 5.3. After this, the

Michaelis-Menten kinetic constants were evaluated from the data obtained. Figure 5.12 shows the hydrolysis of paraoxon with paraoxonase 1 (G3C9).

O O PON1 + O P O NO2 O P OH HO NO2 H3C H3C O O 412 nm

CH3 CH3

Figure 5.12. Paraoxon hydrolysis with PON1

The reactions were carried out in triplicates and the data obtained from the absorbance of p-nitrophenol with time for two runs is shown in Table 5.3. This table also shows the calculated the initial velocities for the formation of each concentration of the substrate to be used for the Michelis-Menten equation fitting.

The data was fitted with the Kaleidegraph graph fitting software. This fitting provided the values of Km, kcat and Vmax for the enzymes. Figure 5.13 shows the fitting of the G3C9 data obtained. The same was done for the PEGylated G3C9 data and the

Michelis-Menten plot is shown in Figure 5.14. The values of Km, kcat and Vmax for the

G3C9 enzyme and the PEGylated G3C9 were calculated and are shown in Table 5.4. For

126

comparison one peak obtained from SEC which contained both was analyzed for the kinetic parameters and are also shown in the Table 5.4.

Table 5.3. UV spectrophotometric data for the appearance of p-nitrophenol from the hydrolysis of paraoxon with G3C9 and calculation or initial velocities PON conc, mM slope 1 Δconc/min slope 2 Δconc/min

0.26 0.68 0.09 0.92 0.12

0.78 2.29 0.29 2.1 0.27

1.3 3.43 0.44 3.4 0.43

1.9 4.41 0.57 4.7 0.61

2.36 5.20 0.673 5.14 0.665

2.6 4.5 0.58 4.9 0.64

From Table 5.4, it is observed that there is a reduction by half of the Kcat for the

PEGylated sample compared to the unPEGylated sample. The sample that contained the mixture of the PEGylated and G3C9 was also used and it had Kcat values that were in between the two.

127

Michelis-Menten Curve, G3C9 0.6

0.5

0.4

0.3

0.2

0.1

0 0 0.5 1 1.5 2 2.5 3 paraoxon (mM)

Figure 5.13. The MichelisD -Menten plot for G3C9

Michelis-menten PEG-G3C9 0.6

0.5

0.4

0.3

0.2

0.1

0 0 0.5 1 1.5 2 2.5 3

paraoxon (mM) Figure 5.14. Michelis-Menten plot for both G3C9 and PEG-G3C9 and the fitting parameters

128

Table 5.4. Kinetics reaction results of G3C9 and PEG-G3C9 -1 -1 -1 Enzyme Vmax, mM/min Km, mM Kcat, min Kcat/Km, mM min

G3C9 1.1  0.2 2.8  0.5 168.130.0 60.0  11.1

PEG-G3C9 0.71  0.04 1.1  0.1 45.4  0.3 42.2  4.2

The Km and Kcat values recorded revealed a reduction in the activity of G3C9 after

PEGylation which is normally expected. This observation is from other previous studies using other therapeutic proteins that have been PEGylated69. However the reduction recorded for the catalytic efficiency was almost three times. After this experiment the samples were tested for the levels of endotoxins. Then endotoxins removal was carried out to FDA acceptable levels.

5.3.5 Endotoxin removal and testing of the G3C9 and PEG-G3C9 samples

After the removal of the endotoxins using the agarose-immobilized polymyxin gel from thermo, the endotoxin level in the PEGylated G3C9 was quantified with the limulus amebocyte lysate test (LAL) using the LAL Assay kit (Genscript Piscataway, NJ, USA ).

Limulus amebocyte lysate, is the most commonly endotoxin detection systems based on

LAL, which is derived from the blood of horseshoe crab, Limulus polyphemus, and clots upon exposure to endotoxin. A sample is considered to be non-pyrogenic if it has concentration of less than 0.5 endotoxin units (EU)/mL. For water, the allowed level is

0.25 EU/mL. The concentration of the endotoxin is measured spectrophotometrically by hydrolyzing the pre-clotting enzyme with chromogenic substrate which releases a yellow- colored substance known as p-nitroaniline. The time required to attain the yellow substance is related to the endotoxin concentration. Then a color stabilizer is added to

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give a blue color and the absorbance reading taken. The results obtained for the

PEGylated sample and the endotoxin standards were obtained and after this, the data was fitted to a linear curve and the sample endotoxin concentration was extrapolated from the curve as shown in Figure 5.15.

Figure 5.15. Spectrophotometric data of endotoxin testing and removal from G3C9 sample. The concentration of the unknown is extrapolated from the graph

After passing the sample through the endotoxin removal gel four times, the concentration of the endotoxins in the sample was found to be 0.021 EU/mL which was within the required 0.5 EU/mL.

130

5.4 Conclusions

This chapter describes the PEGylation of G3C9, a paraoxonase 1 enzyme variant which is a catalytic Bioscavenger against organophosphate compounds for example nerve agents. The aim of the study was to modify the enzyme with PEG which will give it increased solubility and also increase the enzyme half-life in serum. The enzyme was successfully PEGylated in phosphate buffer pH 8.8 and the PEGylated sample generally had between two to five PEGS. The PEGylated sample was purified with anion exchange chromatography and the pure sample kinetic studies carried out in comparison with the

G3C9. In the kinetic studies, the samples were used to hydrolyze paraoxon. The Kcat/Km values for the PEGylated G3C9 did not reduce significantly. G3C9 had 60.0 mM-1min-1 while the PEGylated had 42.2  4.2 mM-1min-1. After this the PEGylated G3C9 and

G3C9 were purified to remove endotoxins so that the samples can be used for PK studies in animal models against nerve agents.

131

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CHAPTER 6 Summary

This dissertation describes the study of glycosylation structures of several proteins expressed in different organisms. The study also includes PEGylation which a cheap form of glycosylation of proteins expressed in E. coli which does not glycosylate proteins.

The first expression system was Chinese hamster ovary (CHO) cells which is mammalian. This is described in the second chapter where the sugar analysis and glycosylation site of a fusion protein of human CD24 glycoprotein expressed in Chinese hamster ovary (CHO) cells. The sugar structural analysis was obtained using MALDI-

TOF-mass spectrometry. The results from the analysis revealed the presence of both N- and O-glycans.

The major sugars on this protein were O-glycans. This is expected due to the fact that it has many potential O-glycosylation sites. The analysis also revealed presence of sialyl-T antigen, Neu5Acα-2,3/6Galβ-1,3GalNAc which was found to be the most abundant glycan. It appears to contribute more than half of the total glycans from CD24.

The theoretical molecular weight of CD24s 30 amino acids is just 5.9 kDa. This means that this O-glycan is attached to several serines and threonines on the peptide backbone to make up to the 28-32 kDa.

The determination of the glycosylation sites on the CD24Fc was carried out after sugar hydrolysis with TFMS followed by protease digestion. Analysis was carried out by

MALDI-TOF and LTQ-orbitrap mass spectrometry. The results revealed the presence of

138

the N-glycosylation site on the Fc region with the observing of the peptide

EEQYNSTYR+ GlcNAc at m/z 1392.3. The peptide was identified due to the presence of the modification from the innermost N-acetylglucosamine of N-glycans that is attached to the asparagine on the peptide backbone.

The study on the O-glycosylation sites on CD24 was however not obtained after chemically deglycosylating the peptide. This was most likely due to the size and poor ionization of the CD24 peptide. However after completely deglycosylating the peptide by treatment with TFMS twice and treatment with chymotrypsin was the part of the peptide observed. This peptide, SSETTGTSSNSSQSTSNSGL, had no modifying markers since all the sugars were hydrolyzed.

In Chapter 3 a Chlamydomonas reinhardtii protein, Fea1 was analyzed for presence of mammalian-like or palnt-like N-glycosylation. Chlamydomonas reinhardtii is a single cell green alga. The first step was by detecting for presence of glycosylation on the protein. The method that was used was glycodetection by chemiluminiscence. The result obtained showed that Fea1 was glycosylated since the Fea1 band was fluorescent active after labeling with a fluorescent tag.

To analyze the types of monosaccharides present on this protein monosaccharide analysis with GC-MS was carried out. The results obtained showed the presence of mannose, galactose and arabinose. However, the presence of N-acetylglucosamine was not observed. This monosaccharide residue is the one that is normally present in mammal

N-oligosaccharide. This therefore means that the oligosaccharides present on this glycoprotein might not be mammalian like N-glycans and higher plants.

139

Release with Peptide-N-Glycosidase A enzyme, revealed presence of some glycans which had similar structure of glycans from plants due to the presence of

Pentoses, hexoses and N-acetyl hexosamines. The structures however were not conclusively derived. However this modification is not plant-like due to the absence of

GlcNAc residue from the monosaccharide analysis with GC-MS.

LTQ MS did not show presence of any of these four potential N-glycosylation sites.

In Chapter 4, the glycoprofiling of glycans released from recombinant TNFR fusion protein is described. TNFR is a protein that is currently used in arthritis treatment in the USA. The market name is Eternercept. This chapter details the experiment in analyzing the glycosylation on this protein that was expressed in the plant Camelina sativa. Expression of therapeutic proteins will be advantageous since they have many advantages as outlined in the introduction of this chapter.

The analysis revealed that the N-glycosylation modification on this protein was plant-like. This is because of the presence of α,1-3 Fucose which was present only when

PNGase A was used but absent when PNGase F was used. PNGase F will not cleave N- glycans with this type of monosaccharide residue attached to the core GlcNAc. Also present was the pentose xylose which is normally present in plant N-glycan.

140

Identification of these sugars is important because their presence on TNFR-Fc will cause immunogenic reaction when injected into humans with their presence.

In the fifth chapter, the PEGylation of G3C9 a recombinant protein of human paraoxon 1 which is a catalytic Bioscavenger against organophosphate compounds for example nerve agents is described. The recombinant protein was expressed in E. coli and purified before PEGylation with a 28 kDa PEG.

The aim of the study was to modify the enzyme with PEG which will give it increased solubility and also increase the enzyme half-life in serum. The enzyme was successfully PEGylated in phosphate buffer pH 8.8 and the PEGylated sample generally had between two to five PEGS. The PEGylated sample was purified with anion exchange chromatography and the pure sample kinetic studies carried out in comparison with the

G3C9.

In these studies, the samples were used to hydrolyze paraoxon to get the kinetic parameters. The Kcat/Km value for the PEGylated G3C9 was almost a third of the non

PEGylated sample. G3C9 had 60.0 mM-1min-1 while the PEGylated had 42.2 mM-1min-1.

After this the PEGylated G3C9 and G3C9 were purified to remove endotoxins so that the samples can be used for PK studies in animal models against nerve agents.

141

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Appendices

Appendix A. O-glycan Chemical Cleavage

I. Ammonia-based β-elimination

1 Dissolve 1 mg of glycoprotein in 28% ammonium hydroxide saturated with

ammonium carbonate at room temperature.

2 Before incubation, add 1 g of ammonium carbonate to this solution and incubate it for

48 h in the dark at 60 oC.

3 Then remove the salts in the solution by lyophilization several times until no salts are

observed in the eppendorf tube.

4 Add about 200 μL of 0.5 M boric acid to the sample and incubate it at 37 oC for 30

minutes and remove the excess borate salts by co evaporated four times with

methanol with a gentle stream of nitrogen gas.

5 Dissolve the remaining sample in 100 μL of de-ionized water and centrifuged for 20

min to precipitate the proteins.

6 Freeze-dry the supernatant, and store at -20 oC for further analysis.

II. β-elimination

1 Dissolve 2 mg of freeze dried CD24Fc in 0.05M NaOH containing 1M sodium

borohydride.

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2 Incubate at 45 oC for 18h.

3 Terminate the reaction by adding glacial acetic acid dropwise until the fizzing stops

(usually 5 drops).

4 Desalt the mixture with a highly acidic resin (amberlyst.)

5 Prepare the resin by loading it in glass micropipette plugged with glass wool to the

constriction point and wash it with methanol and 5 % acetic acid until clear. Put a clip

at the end of column to control flow.

6 After washing with 10 mL acetic acid, load the sample onto the top of column.

7 Allow the sample to run into column and close clip as sample is all loaded.

8 Add 5 % acetic acid to column, open the clip and collect the eluate about 5 mL

9 Freeze dry and the freeze dried sample was dissolved in 0.5 mL 10% acetic acid in

methanol and excess borates evaporated under a stream of nitrogen gas three times.

10 Store at -20 °C for further analysis.

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Appendix B. CD24Fc fragmentation with papain

Pilot fragmentation of IgG

1. Pipette 100μl of a10mg/ml IgG in PBS into 6 eppendorf tubes.

2. Prepare two Papain solutions in digestion buffer one 0.1mg/mL and 0.02 mg/mL.

3. Prepare the digestion buffer was by making 0.02M EDTA (disodium salt) and 0.02 M

cysteine in PBS buffer pH 7.2.

4. To tubes 1 and 4 add 100 μL of the 0.1 mg/mL Papain; 2 and 5 add 100 μL 0.02

mg/mL; and to 3 and 6 the 100μl buffer was added.

5. Incubate the tubes in a circulating water bath at 37 oC for 1h for tubes 1, 2 and 3 and

for 1h for tubes 4, 5 and 6.

6. Stop each reaction by adding 20 mL of 0.3M iodoacetamide in the PBS buffer and

vortex briefly.

7. Monitor the cleavage of the IgG with non-reducing SDS PAGE.

8. After identifying the optimum conditions apply to CD24Fc.

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Appendix C. Papain digests purification with protein A

1. Prepare the protein A by mixing 0.5 g of protein A with buffer A (prepared by

adjusting 0.02 M NaH2 PO4 + 0.15 M NaCl to pH 8.0) and allow to stand for ~45

minutes.

2. Make a 1:1 mixture of this mixture with buffer A was and pour into the column.

3. Allow the solvent to drain while the resin settles.

4. Wash the column with 20 CVs (column volumes) of buffer A.

5. Load the papain fragmented protein sample and use 10 CVs of buffer A to elute the

Fab components from the column and collect four Fractions of 0.5 mL and test with

SDS PAGE.

6. Elute the Fc components with 3 CVs of buffer B (prepared by mixing 25.7 mL 0.2M

Na2H PO4, 24.3 mL 0.1 M citric acid and 50.0 mL deionized water.) and in this case

also collect four fractions and monitor as above.

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Appendix D. Digestion with Enzymes

I. Protein digestion with PGase F

1. Dissolve 200 µg of glycoprotein in 20mM ammonium bicarbonate pH 8.0 (0.9

mL/1mg protein).

2. To this solution add the denaturalization solution was added (100 µL/1 mg protein)

(0.2% SDS containing 100 mM 2-mercaptoethanol).

3. Heat the solution mixture for 15 minutes in boiling water to denature the glycoprotein.

And allpw it to cool.

4. Add PNGase F (10U/1 mg protein) to this reaction mixture and incubate at 37 0C for

24 h.

5. Take 10µl was before adding the enzyme and after incubation to monitor the cleavage

of the N-glycans with SDS-PAGE.

6. Add 20µl of ice cold ethanol to the lyophilized protein to precipitate the protein.

7. Centrifuge the mixture at 13200rpm for 30 min. and recover the glycans in the

supernatant and dry by speed vacuum followed by storage at -20 0C.

II. Protein digestion with PGase A

1. First digest 1 mg protein mixture with trypsin.

2. Stop the reaction by boiling in water for five min and then freeze dry the sample.

3. Dissolve the dried sample 100 mL citrate-phosphate buffer pH 5.0.

4. Add 0.5 mU of the PNgase A enzyme and incubate the mixture for 24 h at 37 ºC.

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5. Desalt the sample with non-porous graphitized carbon column for MS analysis.

III Trypsin in-solution digestion

1. Dissolve the protein in 1 mL 50 mM ammonium bicarbonate containing 0.1 % SDS

and 4 mM DTT.

2. Heat the solution in boiling water for 15 min. and cool.

3. Add 55 mM of iodoacetamide to this solution and incubate in the dark at room

temperature for 30 minutes.

4. Then add 1:30 of trypsin (enzyme:protein) and incubate overnight at 37 °C.

5. Desalt with C-18 zip-tip for MS analysis.

IV. In-gel trypsin digestion

1. Cut the band from the SDS-PAGE gel and cut into small pieces of 1 mm2.

2. Wash overnight with 50 % MeOH in 10% HAc and once more for 3h.

3. Wash the gel pieces with 3× 25 mM NH4HCO3 in 50 % acetonitrile and dry in a

SpeedVac.

4. For reduction, add 40 µL of 10 mM DTT to the dried gel pieces and incubate for 45

min at 60 ºC.

5. Remove the supernatant and then add 40 µL of 55 mM iodoacetamide in 25 mM

NH4HCO3 and incubated at room temperature for 45 min in the dark.

6. Remove the supernatant and wash with 100 µL 25 mM NH4HCO3 for 10 min and

then dehydrate by vortexing for 5 min with 2×400 µL 25 mM NH4HCO3 in 50 %

acetonitrile and the remove the supernatant.

7. Dry the gel pieces in the speed vacuum and add trypsin in a 1:30 ratio.

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8. Add enough 25 mM NH4HCO3 buffer to cover the gel pieces and incubate at 37 ºC

overnight.

9. Recover the peptides by vortexing with 40 µL 25% acetonitrile in 2.5% TFA, then

0.1 % TFA and finally acetonitrile each time for ten minutes.

10. Combined the extracts and concentrate in a SpeedVac before analyzed by MALDI

after mixing with matrix (2-cyano-4-hydoxycinnamic acid).

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Appendix E. Desalting glycans with non-porous graphitized carbon column

1 Wet the column with 2 column volumes of HPLC methanol.

2 Wash the column with three CVs of 80 % acetonitrile in water (v/v) with 0.1 % (v/v)

trifluoroacetic acid, followed by three CVs of 0.1% TFA or de-ionized water.

3 Dissolve the sample a small amount of water or 0.1% TFA.

4 Load the sample into the column.

5 Desalt with 5 mL of ultra-pure water or 0.1 % TFA.

6 Elute neutral N-glycans with 2 mL of 25 % (v/v) acetonitrile in HPLC grade water

and collect eluate.

7 Elute the acidic N-glycans with 2 mL of 25 % (v/v) acetonitrile in water with 0.05 %

(v/v) trifluoroacetic acid and collect eluate.

8 Freeze-dry the collected eluents and stored at -20 °C.

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Appendix F. Permethylation with cartridges

1 Using a mortar and pestle grind NaOH pellets and place the powder in a 0.8mL spin

column.

2 Wash the column several times with DMSO.

3 Mix the glycans 1-50 ug with 50 uL DMSO, 0.3 uL DI water and 25 uL methyl

iodide.

4 Introduce this mixture immediately into the column and centrifuge at 1000 rpm for 1

minute and collect the flow through in an eppendorf tube.

5 Recover the permethylated sample from the collected sample by extracting thrice

with chloroform.

6 Wash the combined chloroform fractions six times to remove any remaining

contaminants.

7 Dry the sample in a speed vacuum and store at – 20 0C for further analysis.

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Appendix G. CD24 deglycosylation with TFMSA

1. Weigh 1 mg into a glass vial and completely dry it in a lyophilizer for 24 h.

2. Cool the dried sample on ice before reaction.

3. Cool Trifluoromethanesulfonic acid (TFMS) on ice and add 150 µL of the pre-cooled

TFMS to the pre-cooled sample reaction vial and then rapidly seal the vial with a cap.

4. Shake the reaction mixture was gently for 5 minutes until the glycoprotein completely

dissolves.

5. Incubate the reaction mixture on ice for 25 minutes more with occasional shaking.

6. Cool 60% Pyridine Solution and the reaction mixture to approximately –15 °C in a

methanol-dry ice bath. Shake occasionally to avoid solidification of the pyridine

solution. Warm it gently in case of solidification until it melts completely.

7. Add 4 µL of the 0.2% Bromophenol Blue Solution as an indicator dye along the

inside wall of the pre-cooled sample reaction vial and mix. (After the addition, the

color of the solution should red).

8. After immediately adding the indicator solution, add the pre-cooled 60% Pyridine

solution dropwise to the sample reaction vial, with mixing and cooling the sample

reaction vial between drops. Maintain the reaction in cold conditions at all times and

avoid its solidification. (In case of precipitation, 20 µL of water was added to

dissolve the precipitate).

9. Continue with the addition of the 60% Pyridine Solution until the color of the solution

changes from red to yellow to light purple or blue. (Carry out the entire process of

171

neutralization quickly, keeping the reaction mixture cold at all stages to minimize

protein degradation).

10. Purify the deglycosylated protein for MS and/or SDS-PAGE analysis by dialyzing

against 20 mM ammonium bicarbonate solution.

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Appendix H. GC-MS of alditols acetates

Hydrolysis of Glycans from Glycoproteins

1 Add 50-100 µl of 2.5 M TFA to 500 µg of dry sample in a 15-ml screw-cap glass tube

with Teflon-lined lid. Flushed the tube with argon, and cap the tube.

2. Hydrolyze the sample by heating the tube for 4 hours at 100-110 ºC on a heating

block and allow the sample to cool to room temperature before opening.

3 Evaporate the TFA with a stream of nitrogen.

Reduction of Monosaccharides to Alditols

1 In this step include a tube containing a dried standard sugar mix.

2 Dissolve the residue in 50 µl of 2 M NH4OH, and add 50 µl of 1 M freshly prepared

sodium borohyride in 2 M NH4OH. Sonicate the sample for 1 minute, and incubate it

for 2.5 hours at room temperature.

3 Destroy the excess sodium borohyride by carefully adding a total of 15 µl of glacial

acetic acid in three equal aliquots. (Ensure that the reaction fizzes upon addition of the

acetic acid).

4 Dry the sample with a stream of nitrogen and dissolve it in 250 µl of 5% acetic acid in

methanol and dry it with a stream of nitrogen.

5 Repeat Step 4 two more times.

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Acetylation of Alditols

1 Add 250 µl of acetic anhydride to the sample and sonicate it for 5 minutes to ensure

that the entire sample is dissolved and then incubated the sample for 2.5 hours at 100

ºC.

2 Destroy the excess acetic anhydride by adding 2 ml of H2O to the sample. Mix it, and

then allow it to stand for 10 minutes at room temperature.

3 Extract the peracetylated sugars twice with 500 µL of chloroform. Vortex well, and

centrifuge to aid phase separation.

4 Combine the extracts and wash them twice with 1-mL aliquots of H2O and transfer the

chloroform phase to a small glass vial, and dried with a stream of nitrogen at room

temperature (Avoid over drying as volatile derivatives could be lost).

5 Redissolve the extracted products in a suitable amount of chloroform and analyze with

GC-MS.

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Appendix I. G3C9 expression

1 Carry out the expression in 1L. Start by growing starter from a single colony

overnight at 37 ºC in 5 mL 2xTY supplemented with 100 µg/mL ampicillin, 15

µg/mL kanamycin and 1 mM CaCl2.

2 Use a 5 mL starter into a 1 L 2xTY containing 100 µg/mL ampicillin, 15 µg/mL

kanamycin and 1 mM CaCl2 and grown for 12h at 28 ºC. Then harvest the cells at

2500g, for 15 minutes.

3 For protein extraction,suspend the solid in lysis buffer (Tris pH 8 50 mM, CaCl2

1mM, NaCl 50 mM and DTT 0.1 mM).

4 Then transfer the cell suspension through a syringe with needle to aid in cell breaking.

5 Sonicate the solution on ice with a large probe for 1 min six times with an intensity of

6 To the sonicated solution, add 0.1 % tergitol NP-10 and shake for 2.5 h at RT.

7 Centrifuge the solution at 10000 rpm for 1h and transferred to a clean tube before Ni

column purification.

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Appendix J. Ni column purification

1 Wash a Ni-column with 50 mL water.

2 Load the sample loaded manually, and wash with 50 mL activity buffer (Tris pH 8 50

mM, CaCl2 1mM, NaCl 50 mM and 0.1 % tergitol NP-10).

3 Wash with 50 mL activity buffer containing 25 mM Imidazole.

4 Wash with 25 mL activity buffer containing 50 mM Imidazole.

5 Lastly the sample was eluted with 50 mL activity buffer containing 250 mM

Imidazole and collect fractions containing sample with activity against paraoxon.

6 Analyze for purity with SDS-PAGE.

176

Appendix K. Anion exchange chromatography

1 Equilibrate the Hitrap Q column with 5 CVs of buffer A at flow rate 2 mL/min.

2 Load the PEGylated sample at a flow rate of 1 ml/min.

3 Carry out the separation with a linear gradient of /0-100 % B (20 mM pH 8 Tris, 1

mM CaCl2, 0.1% Tergitol, 1M NaCl) in buffer A (B without NaCl) , Flow rate 2

mL/min.

4 Collect 3-mL fractions and ascertain purity with SDS-PAGE.

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Appendix L. Kinetic activity Measurements

1 Make up a fresh batch of 0.13 M Paraoxon (in 100% MeOH) working stock on the

day of experiment.

2 Set up the Spectrophotometer (SoftMax Pro V5) and monitor reactions Kinetics at

OD 412 for 10 minutes reaction duration at 20 seconds read interval.

3 Determine the slope from linear portion by adjusting “Lag and End” times.

4 Setup the reaction by adding reagents in the following order to well-plate: Reaction

Buffer; Paraoxon and 10 µL of enzyme for a total of 200 uL as summarized below:

Vol. of Vol. from 0.13 M Effective Total Reaction Working [Paraoxon] Buffer (µL) Paraoxon Stock (µL) in Rxns (mM) 189.6 0.4 0.26 188.6 1.2 0.78 188.0 2.0 1.30 187.0 3.0 1.90 186.4 3.6 2.36 186.0 4.0 2.60 5 Then record the measured slope (change in absorbance per minute due to product

formation) of the reaction.

178

Appendix M. Endotoxin removal

1 First, regenerate the Detoxi-Gel Resin by washing with five resin-bed volumes of 1%

sodium deoxycholate, followed by 3-5 resin-bed volumes of pyrogen-free buffer (20

mM Tris pH 8.0, 1 mM CaCl2,) to remove the detergent. Regenerate the resin before

each use.

2 Equilibrate the resin with 3-5 resin-bed volumes of pyrogen-free buffer.

3 Apply the sample to the column and aliquots of pyrogen-free buffer and collected the

flow-through. (With a gravity flow column, the sample begins to emerge from the

column about 90% of the bed volume has been collected). For greater efficiency, stop

the column flow after sample has entered the resin bed, and incubate the column for

one hour before elution.

179

Appendix N. Figures

1200000

66250 Intensity

33213 <35658> <36993> <47338> <23268><24048><24543> <45826><48553> <24497><24801><25706> <48835><49523> 0 30000 m/z 110000 C:\Documents and Settings\Wang Group\Desktop\transfers from chem dept\Chem-MALDI\CD24 protein\TFMS\71709\after oxd elimination\0_A7\1\1SLin\pdata\1\1r (23:03 04/19/10) Description: Appendix N.1 MALDI-TOF-MS spectra of CD24Fc after TFMS reaction and sample not reduced with DTT

180